WO2025076141A1

WO2025076141A1 - Viral delivery of grna to the scion

Info

Publication number: WO2025076141A1
Application number: PCT/US2024/049665
Authority: WO
Inventors: Rosa BARROCO; Janhenk BOEKELOO; Frédéric VAN EX; Pauline LANNOO; Katalin Toth; Michael Lee NUCCIO; Palak KATHIRIA; Yi Jia; Anna DANEVA
Original assignee: Inari Agriculture Technology Inc
Current assignee: Inari Agriculture Technology Inc
Priority date: 2023-10-03
Filing date: 2024-10-02
Publication date: 2025-04-10
Anticipated expiration: 2026-04-03
Also published as: EP4554371A1

Abstract

The present disclosure provides methods for editing a genomic target in a soybean plant through the use of viral-mediated delivery of at least one genome editing reagent. Also provided are methods of editing a grafted scion by expression in the rootstock of a meristem transport segment- tagged endonuclease, and delivery of additional genome editing reagents to the plant or to the scion by virus-mediated delivery. Also provided are viral vector systems for use in the methods herein, and soybean plants, seeds, and cells edited using the provided methods.

Description

VIRAL DELIVERY OF GRNA TO THE SCION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/587,514, filed October 3, 2023; and of U.S. Provisional Application No. 63/640,823, filed April 30, 2024; both of which are hereby incorporated by reference in their entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (165362001740seqlist.xml; Size: 138,231 bytes; and Date of Creation: October 1, 2024) is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0003] In some aspects, the present invention relates to gene editing methods in soybeans that use virus-mediated delivery of guide RNAs for Cas enzymes. In some aspects, the Cas enzymes that are fused to a meristem transport segment and can be transported from the root to the meristem of the plant, and in other aspects, the Cas enzymes are delivered by virus-mediated delivery. In some aspects, the present invention relates to gene editing methods in plants that use Cas enzymes that are fused to a meristem transport segment and can be transported from the root to the meristem of the plant, and virus- mediated delivery of guide RNAs for the Cas enzymes.

BACKGROUND

[0004] 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. Editing genes in plant meristem cells can give rise to edited germline cells and, ultimately, edited seed to produce edited plants without the requirement for lengthy and difficult tissue culture and plant regeneration.

[0005] Meristem nuclei can also be edited without the introduction of a transgene to the meristem itself. 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. Sci Rep 2011, 1: 73; Jackson and Hong Front Plant Sci 2012: doi: 10[dot]3389/fpls[dot]2012[dot]00127). Similar results have been shown for sequences derived from some transfer-RNAs (Zhang et al. Plant Cell 2016, 28: 1237-1249). Meristem transport segments have been fused to genome editing reagent transcripts to enable them to move from one part of the plant, such as the leaf or root, to the shoot apical meristem (Doyle et al. BioRxiv, 2019: 805036). Thus, the 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. Heritable edits are the result. However, this method is still limited to species that are amenable to transformation.

[0006] 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. Plant Cell 2016, 28: 1510-1520) or biolistics (Kikkert et al. Methods Mol Biol Clifton NJ 2005, 286: 61-78). Species of plants that are difficult to transform are difficult to edit in this manner, and the introduction of a transgene in order to make the edits requires additional screening and/or breeding to later remove the transgene or ensure that it does not cause unwanted effects by disrupting an existing genomic element.

[0007] 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.

[0008] 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.

[0009] One important crop plant that has been especially recalcitrant to VIGE is soybean (Glycine), even though engineered plant viruses have served soybean functional genomics research for years. The application of both Virus-Induced Gene Silencing (VIGS) and Overexpression (VOX) have been proved instrumental in the elucidation of soybean genes implicated in disease resistance (Liu, J-Z. et al. Gaining insight into soybean defense responses using functional genomics approaches, Briefings in Functional Genomics, Volume 14, Issue 4, July 2015, Pages 283-290) and growth habit (Liu B. The soybean stem growth habit gene Dtl is an ortholog of Arabidopsis TERMINAL FLOWER 1. Plant Physiol. 2010 May; 153(1): 198-210). However, while VIGS has been applied for functional genomic studies in soybean, to date no reports have addressed virus-induced gene editing in this economically important crop. Popular soybean-specific viral vector systems offer low cargo capacity, which makes it challenging to engineer Cas-carrying variants. Drawing from comparable methods in different plant species, creating stable transgenic soybean lines that continuously express an editing enzyme could address this issue. However, these lines might not be easily obtainable for many genotypes.

[0010] A recent patent application (U.S. Patent Application No. 2022/0090107) describes an alternative virus - Tobacco Ringspot Virus (TRSV) from the picornavirus family- which was engineered to carry a Cas9-specific gRNA molecule fused to the viral coat protein. Engineered viral vectors were first inoculated to tobacco plants to raise infectious inoculum, which was then applied to a soybean shoot culture or to soil-grown soybean plants. Another study reported the use of modified Apple Latent Spherical Virus (ALSV), which typically infects soybeans, to transport and release gRNAs using the endoribonuclease Csy4 (Luo, Y. et al. Development of a Csy4-processed guide RNA delivery system with soybean-infecting virus ALSV for genome editing. BMC Plant Biol. 2021. 21, 419). Successful Cas9-mediated genome editing was first demonstrated in tobacco (Nicotiana benthamiana) by editing both the native phytoene desaturase (PDS) loci and the introduced 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS) sequence in using Luo et al.’s developed “Cas9- based Csy4-processed ALSV Carry” (CCAC) system. Additionally, when applied to in-vitro grown soybean hairy roots, the CCAC vectors induced mutations in two soybean paralogs of rice gene RING- type E3 ubiquitin ligase (GW2). Notably, none of these documented cases of soybean VIGE successfully induced heritable mutations.

[0011] A need exists in the art for a method of introducing heritable edits to the meristem of a plant without the introduction of a transgene to the meristem genome and with the possibility of editing a multitude of species. Consequently, a need persists in the art for a method of introducing heritable edits to the meristem of a soybean plant without the introduction of a transgene to the meristem genome and without requiring transformation. This disclosure answers this need by providing a system based on a soybean-infecting virus that can induce both somatic and inheritable mutations. SUMMARY

[0012] One aspect of the present disclosure provides a method of editing a genomic target in a meristem cell of a soybean plant comprising: a) delivering a guide RNA (gRNA) directed to the genomic target to the meristem cell in the soybean plant by virus-mediated delivery; and b) delivering a Cas nuclease to the meristem cell, wherein the Cas nuclease and the guide RNA edit the genomic target in the meristem cell of the soybean plant, thereby editing the genomic target in the meristem cell. In some embodiments, the method further comprises: c) allowing the meristem cell to generate a seed comprising the edited genomic target; and d) collecting the seed.

[0013] Another aspect of the present disclosure provides a method of producing a soybean seed comprising an edited genomic target, the method comprising: a)delivering a guide RNA (gRNA) directed to the genomic target in a meristem cell of a parent soybean plant by virus-mediated delivery; and b) delivering a Cas nuclease to the meristem cell of the parent soybean plant, wherein the Cas nuclease and the guide RNA edit the genomic target in the meristem cell of the parent soybean plant, and wherein the meristem cell produces a soybean germline cell that contributes to the soybean seed, and thereby producing the soybean seed comprising the edited genomic target.

[0014] In some embodiments, which may be combined with any preceding embodiment, the edited genomic target is inherited by at least one progeny or seed of the soybean plant. In some embodiments, which may be combined with any preceding embodiment, virus-mediated delivery comprises using a viral vector comprising the gRNA. In some embodiments, which may be combined with any preceding embodiment, the Cas nuclease is fused to a meristem transport segment (MTS). In some embodiments, which may be combined with any preceding embodiment, the virus-mediated delivery comprises infecting the soybean plant with an inoculum comprising the viral vector.

[0015] In some embodiments, which may be combined with any preceding embodiment, the viral vector comprises a recombinant plant virus that comprises the guide RNA or a polynucleotide encoding the guide RNA. In some embodiments, the recombinant plant virus used in the virus- mediated delivery is a virus with a segmented genome. In some embodiments, which may be combined with any preceding embodiment, the recombinant plant virus further comprises an expression cassette comprising an endogenous visible marker gene or a reporter gene, optionally wherein the reporter gene encodes a fluorescent reporter. In some embodiments, which may be combined with any preceding embodiment, the recombinant plant virus is capable of cell-to-cell movement.

[0016] In some embodiments, which may be combined with any preceding embodiment, wherein the viral vector comprises bean pod mottle virus (BPMV), optionally wherein the BPMV vector comprises BPMV-RNA2 and/or BPMV-RNA1. In some embodiments, the BPMV-RNA2 and/or BPMV-RNA1 is linked to, or otherwise carries, the gRNA. In some embodiments, which may be combined with any preceding embodiment, the viral vector is delivered to a leaf, shoot, stem, root, or other vegetative tissue. In some embodiments, which may be combined with any preceding embodiment, the viral vector comprises at least two gRNAs, at least three gRNAs, at least four gRNAs, at least five gRNAs, at least six gRNAs, at least seven gRNAs, or at least eight gRNAs, optionally wherein each gRNA is directed to: (i) a different genomic target in the soybean plant; or (ii) a same genomic target in the soybean plant.

[0017] In some embodiments, which may be combined with any preceding embodiment, the gRNA is directed to a regulatory or coding sequence, optionally wherein the regulatory or coding sequence contributes to a trait selected from the group consisting of: photosynthetic ability or efficiency; yield or fertility; seed number; disease or pest resistance; herbicide or pesticide tolerance; abiotic stressor tolerance; fruit morphology; fruit nutrition; fruit ripening; number of seeds per pod; and leaf size. In some embodiments, which may be combined with any preceding embodiment, infecting the soybean plant comprises inoculating the soybean plant’s leaves, shoot, stem, roots, or other vegetative tissue with a composition comprising the viral vector that contains the gRNA.

[0018] In some embodiments, which may be combined with any preceding embodiment, the Cas nuclease is delivered by virus-mediated delivery. In some embodiments, which may be combined with any preceding embodiment, the viral vector comprises the Cas nuclease. In some embodiments, which may be combined with any preceding embodiment, RNA encoding the gRNA and/or the Cas nuclease is delivered to the meristem cell of the soybean plant by transport from another plant tissue. In some embodiments, RNA encoding the Cas nuclease is translated in the meristem cell.

[0019] In some embodiments, which may be combined with any preceding embodiment, the viral vector comprising the gRNA further comprises an RNA-guided nuclease. In some embodiments, which may be combined with any preceding embodiment, the viral vector comprising the gRNA further comprises the Cas nuclease. In some embodiments, which may be combined with any preceding embodiment, the Cas nuclease is delivered to the meristem cell of the soybean plant in a second viral vector comprising the Cas nuclease. In some embodiments, which may be combined with any preceding embodiment, the method further comprises infecting the soybean plant with a plurality of viral vectors, wherein each viral vector comprises one or more gRNA and/or the Cas nuclease. In some embodiments, the plurality of viral vectors co-infect the soybean plant simultaneously or infect the soybean plant in more than one round of infection. In some embodiments, which may be combined with any preceding embodiment, the soybean plant overexpresses the Cas nuclease.

[0020] In some embodiments, which may be combined with any preceding embodiment, the genomic target is in a scion. In some embodiments, the Cas nuclease is delivered to the scion by transport from a grafted rootstock. In some embodiments, the method, further comprises transforming the rootstock with a nucleic acid encoding the Cas nuclease prior to grafting. In some embodiments, which may be combined with any preceding embodiment, the scion and the rootstock are the same plant species or different plant species, optionally wherein the rootstock is canola, alfalfa, corn, oat, sorghum, sugarcane banana, or wheat. In some embodiments, which may be combined with any preceding embodiment, the Cas nuclease is delivered by transport from another part of the plant through the plant vascular system. In some embodiments, the inoculum is infectious lysate, optionally wherein the infectious lysate is provided by (a) performing leaf infiltration of soybean or non-soybean leaves with bacteria comprising an infectious cDNA plasmid, and (b) collecting infectious lysate from the soybean or non-soybean leaves, thereby providing the infectious lysate. In some embodiments, the infectious lysate is provided by inoculating a set of first host plants with at least one infectious cDNA plasmid and collecting infectious lysate from the first host plant, the cDNA plasmid comprising: i. the recombinant plant virus; ii. the gRNA; and/or iii. the Cas nuclease. In some embodiments, the method further comprises: a) selecting from the set of first host plants a selected plant that is highly infected with intact viral cargo comprising the recombinant plant virus, the gRNA, and/or the Cas nuclease; b) raising the selected plant; and c) collecting the infectious lysate from the selected plant, the method optionally comprising inoculating a second host plant or a set of second host plants with the infectious lysate from the first selected plant. In some embodiments, the selected plant is identified by (a) detecting expression levels of viral coat protein, and/or detecting the presence of intact viral cargo, wherein detecting the presence of intact viral cargo optionally comprises sequencing infectious cDNA in the first host plant; and/or (b) measuring levels of viral coat protein-encoding mRNA in the first host plant by RT-qPCR.

[0021] In some embodiments, which may be combined with any preceding embodiment, the first host plant or selected plant is Nicotiana or soybean. In some embodiments, which may be combined with any preceding embodiment, the virus-mediated delivery comprises direct leaf rub inoculation with infectious lysate comprising the gRNA. In some embodiments, which may be combined with any preceding embodiment, the gRNA comprises a 5 -methylcytosine group.

[0022] In some embodiments, which may be combined with any preceding embodiment, the viral vector comprises: i. a first ribozyme sequence; ii. a direct repeat; iii. a spacer sequence complementary to the gene of interest; and iv. a second ribozyme sequence, wherein the first ribozyme sequence and the second ribozyme sequence are the same or different sequences. In other embodiments, which may be combined with any preceding embodiment, the viral vector comprises: i. a first direct repeat; ii. a spacer sequence complementary to the gene of interest; and iii. a second direct repeat. In further embodiments, which may be combined with any preceding embodiment, the viral vector comprises: i. a RNase-resistant caged truncated pre-tRNA-like crRNA (catRNA); ii. a first direct repeat; iii. a spacer sequence complementary to the gene of interest; and iv. a second direct repeat. In some embodiments, which may be combined with any preceding embodiment, the gRNA is processed by a ribozyme selected from the group consisting of a hammerhead ribozyme, a hepatitis delta virus (HDV) ribozyme, a Csy4, and a tRNA-derived sequence.

[0023] In some embodiments, which may be combined with any preceding embodiment, the method further comprises screening the soybean plant for viral infection, said screening comprising a visual assessment of the soybean plant for a desired phenotype. In some embodiments, which may be combined with any preceding embodiment, the soybean plant further comprises a nucleic acid encoding a detectable marker fused to a nucleic acid encoding the MTS, optionally wherein the nucleic acid encoding the MTS is located 3’ or 5’ of a nucleic acid encoding the Cas nuclease.

[0024] In some embodiments, which may be combined with any preceding embodiment, 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, optionally 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. In some embodiments, the TLS comprises the nucleotide sequence set forth in SEQ ID NO: 29 or 30.

[0025] In some embodiments, which may be combined with any preceding embodiment, the nucleic acid encoding the Cas nuclease is linked to a promoter, optionally wherein the promoter is active in roots and/or phloem companion cells. In some embodiments, the promoter is a constitutive promoter, optionally wherein the constitutive promoter is a ubiquitin promoter. In some embodiments, the promoter is selected from the group consisting of a promoter from a Arabidopsis WRKY6 gene, a promoter from a chickpea WRKY31 gene, a promoter from a carrot MYB113 gene, a promoter from a corn GLU1 gene, a promoter from a strawberry RB7-type TIP-2 gene, a promoter from a banana TIP2-2 gene, 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 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 JMJ 18 promoter, and a phloem protein 2 (PP2) promoter, or the promoter of an orthologous gene thereof.

[0026] In some embodiments, which may be combined with any preceding embodiment, the Cas nuclease is codon-optimized for expression in dicots. In some embodiments, which may be combined with any preceding embodiment, the Cas nuclease is codon-optimized for expression in soybean. [0027] In some embodiments, which may be combined with any preceding embodiment, the Cas nuclease is a nuclease is selected from the group consisting of Cas9, Casl2a (Cpfl), Casl2e (CasX), Casl2d (CasY), C2cl, C2c2, C2c3, Casl2h, Casl2i, and a Mini Cas. In some embodiments, which may be combined with any preceding embodiment, the Cas nuclease is a Cas nickase, optionally wherein 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, which may be combined with any preceding embodiment, the gRNA is heterologous to the soybean plant. In some embodiments, which may be combined with any preceding embodiment, the gRNA and the Cas nuclease form a complex and introduce a single- or double-stranded break in the sequence of the genomic target. [0028] In some embodiments, which may be combined with any preceding embodiment, the method further comprises delivering a donor template DNA to the soybean plant, optionally wherein the donor template DNA is delivered by infecting the soybean plant with a viral vector that infects the meristem cell. In some embodiments, a sequence from the donor template DNA is incorporated into the genome of the soybean plant. In some embodiments, the sequence from the donor template DNA is incorporated into the genome of the soybean plant at the genomic target. In some embodiments, which may be combined with any preceding embodiment, the donor template DNA is delivered to the soybean plant using the same viral vector as the gRNA. In some embodiments, which may be combined with any preceding embodiment, the Cas nuclease is fused to a reverse transcriptase. In some embodiments, which may be combined with any preceding embodiment, the guide RNA comprises at its 3’ end a priming site and an edit to be incorporated into the genomic target. In some embodiments, which may be combined with any preceding embodiment, the gRNA is a prime editing guide RNA (pegRNA). In some embodiments, which may be combined with any preceding embodiment, the donor template DNA is delivered to the soybean plant using a different viral vector than the viral vector comprising the gRNA. In some embodiments, which may be combined with any preceding embodiment, the donor template DNA confers a desired trait on the plant, and optionally wherein the donor template comprises an exogenous or endogenous sequence.

[0029] In some embodiments, which may be combined with any preceding embodiment, the virus- mediated delivery comprises transforming the root of the plant with a bacterium comprising a binary vector comprising a recombinant plant virus comprising the guide RNA or a nucleic acid encoding the guide RNA, optionally wherein the bacterium further comprises a binary vector comprising a nucleic acid encoding the Cas nuclease. In some embodiments, which may be combined with any preceding embodiment, the meristem cell is in a shoot apical meristem or an axillary meristem.

[0030] In some embodiments, which may be combined with any preceding embodiment, the editing of the genomic target results in the increased expression of a gene of interest in the soybean plant, wherein the genomic target inhibits the gene of interest when expressed in a control plant. In some embodiments, which may be combined with any preceding embodiment, the genomic target is involved in viral defense, Non-Homologous End Joining (NHEJ), Mismatch Repair (MMR), or condensing chromatin. In some embodiments, which may be combined with any preceding embodiment, the method is preceded by delivering a gene or gene fragment to repress viral defense, inhibit Non-Homologous End Joining (NHEJ), inhibit Mismatch Repair (MMR), arrest cells in the S or G2 phases, or suppress enzymes that condense chromatin. In some embodiments, delivering the gene or gene fragment comprises Agrobacterium-mediated transformation or rub-inoculation. In some embodiments, which may be combined with any preceding embodiment, the gene or gene fragment is selected from the group consisting of double-stranded RNA-binding protein (DRB) genes, the DRB4 gene, genes for Dicer-like proteins, HEN1 (HUA ENHANCER 1) methyltransferase gene, and the SGS3 gene. [0031] Also provided in the present disclosure is a viral vector system for use in soybean editing, the system comprising: i. a plant virus genome component; ii. one or more gRNA; and iii. a direct repeat and/or self-cleaving ribozyme sequence flanking the one or more gRNA.

[0032] Also provided in the present disclosure is a bean pod mottle virus (BPMV) viral vector system comprising: i. a BPMV genome component; ii. one or more gRNA; and iii. a direct repeat and/or self-cleaving ribozyme sequence flanking the one or more gRNA.

[0033] Also provided in the present disclosure is a viral vector system for use in soybean editing, the system comprising: i. a plant virus genome component; ii. one or more pegRNA; and iii. a Cas nuclease fused to a reverse transcriptase (RT).

[0034] Also provided in the present disclosure is a viral vector system for use in soybean editing, the system comprising: i. a plant virus genome component; ii. a guide RNA (gRNA) directed to a genomic target in soybean; and iii. a Casl2f nuclease.

[0035] Also provided in the present disclosure is a viral vector system for producing an edited genomic target in a soybean plant, the system comprising: a) a plant virus genome component; b) a guide RNA (gRNA) directed to the genomic target; and c) a Cas nuclease expressed in the meristem cell of the soybean plant.

[0036] Another aspect of the present disclosure provides a method of using a bean pod mottle virus (BPMV) to edit a genomic target in a soybean plant comprising: a) infecting the soybean plant with a recombinant bean pod mottle virus (BPMV) carrying a guide RNA (gRNA) directed to the genomic target; b) expressing a Cas nuclease in the plant; c) allowing sufficient time to elapse for the virus to infect meristem cells in the soybean plant; and thereby using the BPMV to edit the genomic target, optionally wherein the gRNA is processed by a ribozyme selected from the group consisting of a hammerhead ribozyme, a hepatitis delta virus (HDV) ribozyme, a Csy4, and a tRNA-derived sequence.

[0037] In some embodiments, which may be combined with any preceding embodiment, the method further comprises screening the soybean plant for successful editing of the genomic target, said screening comprising: a) visually assessing the soybean plant for at least one desired phenotype; and/or b) sequencing nucleic acid of cells produced by the meristem cell after delivery of the BPMV vector.

[0038] Another aspect of the present disclosure provides a method for producing a soybean meristem cell having an edited genomic target, the method comprising: a) delivering a viral vector carrying a gRNA to a soybean meristem cell, wherein the soybean meristem cell comprises a Cas nuclease; b) allowing the gRNA and the Cas nuclease to modify the soybean meristem cell; and c) thereby producing the soybean meristem cell having the edited genomic target.

[0039] Another aspect of the present disclosure provides a method of editing a genomic target in a soybean plant scion comprising: grafting the scion onto a rootstock comprising a Cas nuclease, wherein the rootstock comprises nucleic acid encoding the Cas nuclease fused to a meristem transport segment (MTS); and delivering a guide RNA for the Cas nuclease to the scion by virus-mediated delivery, optionally wherein the scion comprises a leaf, a shoot, a stem, or other vegetative tissue. In some embodiments, the method further comprises transforming the rootstock with nucleic acid encoding the Cas nuclease prior to grafting. In some embodiments, which may be combined with any preceding embodiment, the method further comprises retrieving a progeny of the scion, wherein the progeny comprises the edited genomic target.

[0040] Also provided in the present disclosure is a soybean plant produced by growing the seed of any preceding embodiment, wherein the produced soybean plant comprises the edited genomic target. [0041] Also provided in the present disclosure is a soybean seed produced by the method of any preceding embodiment, wherein the produced soybean seed comprises the edited genomic target.

[0042] Also provided in the present disclosure is a soybean meristem cell produced by the method of any preceding embodiment, wherein the soybean meristem cell comprises the edited genomic target.

[0043] Also provided in the present disclosure is a kit comprising the viral vector system of any preceding embodiment and an instruction manual for using the kit.

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIGS. 1A-1B show editing results of targeting PDS in soybean plants. FIG. 1A shows the mottled-leaf phenotype associated with BPMV infection and the bleached phenotype associated with inactivation of phytoene desaturase (PDS) (left, “BPMV_PDS”), contrasted against the wild-type soybean plants infected with BPMV but still producing functional PDS protein (right, “BPMV_WT”). FIG. IB shows the bleached phenotype of bean pods of soybean plants that have been successfully edited to inactivate PDS.

[0045] FIG. 2 shows a sequence alignment of polymerase chain reaction (PCR) amplicon regions for PDS 18 (above) and PDS 11 (below) used for genotyping of PDS edits (215 -nucleotide fragments). These two fragments differ only in three nucleotides (1=A48C; 2= C51T; 3=G195A).

[0046] FIG. 3 shows a schematic of a gRNA fused by flanking tRNA spacers. “BPMV-RNA2 WT” represents an isolated RNA2 genome portion of the bean pod mottle virus (BPMV); “tRNA” represents the tRNA spacers functioning as tags for processing; “DR” represents a direct repeat; “AAAAAA” represents a poly-A tail; each color of spacer represents the spacer sequence of a different gRNAs, and they may or may not have the same sequence. A “spacer sequence” is 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 site or gene of interest. DETAILED DESCRIPTION

[0047] All references cited herein are hereby incorporated by reference in their entirety.

Definitions

[0048] The use of the terms “a” and “an” and “the” and “at least one” and similar language in the context of describing embodiments of the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted.

[0049] The phrase “allelic variant” as used herein refers to a polynucleotide or polypeptide sequence variant that occurs in a different strain, variety, or isolate of a given organism.

[0050] 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. Thus, 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). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0051] As used herein, 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.

[0052] As used herein, 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. In some examples, 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. Given the complementary pairings of nucleotide bases, the nucleotide sequences of the present disclosure should be understood to include their complementary sequences.

[0053] As used herein, the term “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.

[0054] As used herein, 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.

[0055] As used herein, the term “CRISPR-Cas nuclease,” “CRISPR Cas nuclease,” and “Cas nuclease” are used interchangeably herein to refer to all RNA-directed nucleases and RNA-guided nucleases.

[0056] As used herein, the term “endogenous” refers to something that can be found in an organism prior to human intervention. An “endogenous sequence” refers to a DNA sequence located in the genome of the organism prior to editing.

[0057] As used herein, the term “engineered” means artificial, synthetic, or not occurring in nature. For example, a polynucleotide that includes two DNA sequences that are heterologous to each other can be engineered or synthesized by recombinant nucleic acid techniques.

[0058] As used herein, the term “exogenous” refers to something that cannot be found in an organism prior to human intervention. An “exogenous sequence” refers to a DNA sequence that is not located in the genome of an organism prior to editing. An exogenous sequence can be an edited sequence, a synthetic sequence, or a sequence from a different organism.

[0059] As used herein, the terms “heritable genetic modification”, “heritable edit”, and “heritable modification” refer to any insertion, substitution, or deletion in the genomic sequence of a plant that is at least present in a meristem cell of the plant, such that at least one progeny of the plant possesses the same altered genomic sequence.

[0060] 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.

[0061] As used herein, the term “heterograft” refers to a graft between a rootstock and a scion of different species.

[0062] As used herein, the term “homograft” refers to a graft between a rootstock and a scion of the same species.

[0063] As used herein, 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. [0064] As used herein, 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.

[0065] As used herein, the phrase “Mini Cas” or “miniature Cas” refers to RNA-guided nucleases with a relative smaller protein size compared to the well-studied CRISPR-Cas nucleases Cas9 and Casl2a. Mini-Cas may be synonymously used with “Miniature nucleases”, “Compact nucleases”, “Hypercompact nucleases”, “Miniature Cas nucleases”, “Miniature Genome Editors”, “Small nucleases” Example include ISDra2, ISYmul, ISAaml, IsDgelO, IsAaml, enlscB, TnpB, IscB and Fanzor.

[0066] As used herein, the terms “modification,” “edit”, and “modify” are used interchangeably herein to refer to any insertion, substitution, or deletion of any number of nucleotides in a genomic sequence.

[0067] As used herein, 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. Similarly, 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.

[0068] As used herein, the term “linked” or “operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, an RNA molecule comprising a “meristem transport sequence” (MTS) is linked to a guide RNA if the MTS provides for delivery of the guide RNA to meristem cells. For instance, an RNA molecule comprising BPMV-RNA2 is linked to a guide RNA if the BPMV-RNA2 provides for delivery of the guide RNA to meristem cells.

[0069] As used herein, the terms “orthologous,” “ortholog,” 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.

[0070] As used herein, the term “plant” includes a whole plant and any descendant, cell, tissue, or part of a plant. The term “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. In contrast, some plant cells are not capable of being regenerated to produce plants and are referred to herein as “non-regenerable” plant cells.

[0071] As used herein, the phrase “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. The phrase “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. In certain embodiments, a substantially purified RNA is at least 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9% free of contaminating compounds by weight. A substantially purified RNA molecule can be combined with other compounds including buffers, RNase inhibitors, surfactants, and the like in a composition.

[0072] As used herein, the term “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. Where a polynucleotide contains double-stranded regions formed by intra- or intermolecular hybridization, the length of each doublestranded 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 doublestranded RNA or single- or double-stranded DNA or double-stranded DNA/RNA hybrids or chemically modified analogues or a mixture thereof. In various embodiments, 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. In embodiments, the polynucleotide includes chemically modified nucleotides (see, e.g., Verma and Eckstein Annu. Rev. Biochem. 1998, 67: 99-134); for example, the naturally occurring phosphodiester backbone of an oligonucleotide or polynucleotide can be partially or completely modified with phosphorothioate, phosphorodithioate, or methylphosphonate internucleotide 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). Modified nucleic acids, particularly modified RNAs, are disclosed in U.S. Pat. No. 9,464,124, incorporated by reference in its entirety herein.

[0073] As used herein, the terms “progeny” or “plant progeny” refer to any zygote, embryo, endosperm, callus, seed, seedling, or second generation of a plant that is produced after a parent plant cell undergoes meiosis and, in some cases, syngamy.

[0074] As used herein, the phrase “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. 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. 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. Of particular interest are 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). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See Mol. Biol., 48: 443-453 (1970).

[0075] As used herein, the phrase “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.

[0076] As used herein, the phrase “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. Plant Physiol 2007, 145(4): 1155-1160); or a ternary vector system wherein the system further comprises an accessory plasmid or virulence helper plasmid comprising an additional virulence gene cluster (Anand et al. Plant Mol Biol 2018, 97(1- 2): 187-200).

[0077] As used herein, the terms “template,” “template sequence,” “donor template,” “donor template sequence,” “trait sequence,” and “donor trait sequence” can all be used to refer to a DNA polynucleotide provided to a nucleus, cell, or plant in combination with other genome editing reagents in order to integrate a DNA sequence from the DNA polynucleotide into the genome of the nucleus, cell, or plant.

[0078] As used herein, the terms “vascular system” or “vasculature” refer to the transport systems within the plant. This includes xylem, phloem, and cambium.

[0079] Unless otherwise stated, 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.

[0080] Where a term is provided in the singular, the inventors also contemplate aspects of the invention described by the plural of that term.

[0081] To the extent to which any of the preceding definitions is inconsistent with definitions provided in any patent or non-patent reference incorporated herein by reference, any patent or nonpatent reference cited herein, or in any patent or non-patent reference found elsewhere, it is understood that the preceding definition will be used herein.

I. Current Challenges in Transgene-Free Editing

[0082] The use of transgenes in plant editing has undesirable risks. Transgene residuals within plants that have been edited can pose off-target risks and significant regulatory concerns across many jurisdictions. In some embodiments, progeny of a plant modified through the present methods of genomic editing do not contain transgenes encoding the reagents for genomic modification.

[0083] Meristems are resistant to gene editing, evolutionarily protecting the meristem’ s pluripotent cells from mutations that could negatively affect all subsequent cells produced. However, advances have been made in targeting meristem cells. Some of these strategies also avoid the incorporation of a transgene into the meristem. As one example, 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). Fusing highly mobile elements, such as the floral stimulus gene Arabidopsis thaliana Flowering Focus T (FT locus), to either the 5 ’end or 3’ end of guide RNA can improve targeting of meristem cells. Similar results have been shown for sequences derived from some transfer-RNAs (Zhang et al. Plant Cell 2016, 28: 1237-1249). Fusing highly mobile elements, such as the floral stimulus gene Arabidopsis thaliana Flowering Focus T (FT locus), to either the 5 ’end and/or 3’ end of guide RNA can improve targeting of meristem cells. Similar results have been shown for sequences derived from some transfer-RNAs. 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).

[0084] However, these methods are still limited to species that are amenable to transformation, as are most current applications of nuclease modification systems such as the clustered regularly interspaced short palindromic repeats (CRISPR)/CAS system for genome editing (e.g. guide RNA/CRISPR RNAs, CAS nuclease and/or other editing components). Most applications still require plant transformation to introduce the single CRISPR RNA (crRNA) or guide RNA (gRNA) and the Cas nuclease, making CRISPR-mediated crop genome engineering dependent on the availability of established protocols for in vitro transformation and regeneration of edited plants. 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. Plant Cell 2016, 28: 1510-1520) or biolistics (Kikkert et al. Methods Mol Biol Clifton NJ 2005, 286: 61-78). Species of plants that are difficult to transform are difficult to edit in this manner, and the introduction of a transgene to make the edits requires additional screening and/or breeding to later remove the transgene or ensure that it does not cause unwanted effects by disrupting an existing genomic element.

[0085] Along with the biological challenges of successful and efficient transformation, the processes of conducting transformation and plant regeneration are costly in terms of time, labor, and monetary value. Genome editing of commercial crops is currently limited by the well-known recalcitrance of elite materials to transformation. 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.

[0086] 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 US2015/0344912Al, and in U.S. Pat. Nos. 8,697,359, 8,771,945, 8,945,839, 8,999,641, 8,993,233, 8,895,308, 8,865,406, 8,889,418, 8,871,445, 8,889,356, 8,932,814, 8,795,965, and 8,906,616. 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. Other 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[dot]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. 2015/0082478 Al and U.S. 2015/0059010A1 and in International Patent Application PCT/US2015/038767 Al (published as WO 2016/007347, claiming priority to U.S. Provisional Patent Application 62/023,246, with U.S. National Phase application U.S. Ser. No. 15/325,116, now published as U.S. Patent Application Publication 2017/0306349).

[0087] VIGE offers a tissue culture-free alternative to plant transformation: edited seeds can be obtained directly from infected plants. The lengthy and labor-intensive process of plant regeneration - a major bottleneck for recalcitrant species - can be bypassed. The widespread adoption of VIGE, however, is currently hindered by technical challenges related to viral delivery, viral vector payload (e.g., the size of typical CRISPR/Cas enzymes exceeds the payload of commonly used recombinant viral vectors), specificity of host-pathogen interactions, and biosafety concerns. Some of other challenges of currently known VIGE technologies include instability of constructs, selection of correct virus (e.g., balancing the requirement of high infectivity needed for efficient editing with errors in molecular readouts caused by the stress response), mosaicism, and limited meristem invasion.

[0088] One strategy for editing meristems is to utilize viral vectors capable of delivering editing systems or editing system components (such as reagents of a CRISPR/Cas system) to meristem cells. Viruses have been engineered as vectors to silence or modulate gene expression in crop plants, and to deliver components of a nuclease modification system. This technique is commonly referred to as virus- induced gene editing (VIGE). Recently, plant viruses from negative strand RNA were engineered to carry large proteins including Cas 9, Cas 12, and even base editors (Liu Q. et al. Engineered biocontainable RNA virus vectors for non-transgenic genome editing across crop species and genotypes. Molecular Plant. 2023. March 6, 16(3): 616-361). Although successful in inducing edits, these viral vectors have not yet been demonstrated to facilitate transgenerational inheritance of the gene modifications.

[0089] In addition to the above challenges, only a few viral species to date have been shown to invade and infect plant meristems. Due to the narrow specificity of host-pathogen interactions, these viruses cannot be broadly applied to all crops (Bradamante, G et al. Under siege: virus control in plant meristems and progeny. Plant Cell. 2021 Aug 31;33(8): 2523-2537). The addition of mobility tags to virally encoded gRNAs was thought to mitigate the issue of viral exclusion from meristems (Ali Z et al. Efficient Virus-Mediated Genome Editing in Plants Using the CRISPR/Cas9 System. Mol Plant. 2015 Aug; 8(8): 1288-91; Ellison, E.E. et al. Multiplexed heritable gene editing using RNA viruses and mobile single guide RNAs. Nat. Plants. 20206: 620-624; Li T, Hu J, Sun Y, Li B, Zhang D et al. Highly efficient heritable genome editing in wheat using an RNA virus and bypassing tissue culture. Mol Plant. 2021 Nov 1; 14(11): 1787-1798). However, Beernink et al. showed that the success of inheritable VIGE seems to be inherent to the viral vector of choice rather than the addition of a mobility tag to a virally encoded gRNA (Beernink B. et al. Impacts of RNA Mobility Signals on Virus Induced Somatic and Germline Gene Editing. Frontiers in Genome Editing. 2022 4: 2673-3439).

[0090] Furthermore, popular CRISPR/Cas9-based gene editing relies on promoter-driven expression of gRNA because unlike Casl2a (Cpfl), Cas9 cannot process its own gRNA arrays (Fonfara, I. et al. The CRISPR-associated DNA-cleaving enzyme Cpfl also processes precursor CRISPR RNA. 2016. Nature 532, 517-521). Most established soybean viral vectors belong to the RNA+ family of viruses with polyprotein expression strategy; they lack viral sub-genomic promoters and only replicate in the cytosol of the host cell. This renders gRNA transcription by host factors impossible because such factors are compartmentalized in the eukaryotic nuclei. Even though processing spacers can be employed to release the gRNA from its “viral carrier”, this requires either the addition of processing spacers or of self-cleaving ribozymes, which might compromise the integrity or replicating viruses.

[0091] Soybean is an important crop plant that has been especially challenging to edit with methods that avoid transgenes. Relevant to the present disclosure, from all engineered viruses that have been successfully developed for VIGS and VOX in soy, vectors derived from Bean Pod Mottle Virus (BPMV) have been the most widely used. BPMV has a bi-partite genome that consists of RNA1 and RNA2, encoding genes for replication and spread throughout the plant host respectively. In the virus’s natural state, both segments of the genome are required for the virus’s life cycle; RNA1 is critical for genome replication, and RNA2 is critical for cell-to-cell movement. Despite BPMV vectors being a known tool for soybean functional genomics, prior to the present invention there are no reported instances in the literature of their use for somatic or inheritable gene editing in soybean.

II. Methods of Editing

[0092] The present application provides methods of editing a genomic target in a meristem cell of a soybean plant comprising delivering a guide RNA (gRNA) directed to the genomic target in the meristem cell in the soybean plant by virus-mediated delivery; and delivering a Cas nuclease to the meristem cell, wherein the Cas nuclease and the guide RNA edit the genomic target in the meristem cell of the soybean plant. In some embodiments, virus-mediated delivery comprises using a viral vector comprising the gRNA. In some embodiments, the viral vector comprises bean pod mottle virus (BPMV).

[0093] The present application also provides methods of using a bean pod mottle virus (BPMV) to edit a genomic target in a soybean plant comprising: infecting the soybean plant with a recombinant bean pod mottle virus (BPMV) carrying a guide RNA (gRNA) directed to the genomic target; expressing a Cas nuclease in the plant; allowing sufficient time to elapse for the virus to infect meristem cells in the soybean plant; and thereby using the BPMV to edit the genomic target, optionally wherein the gRNA is processed by a ribozyme selected from the group consisting of a hammerhead ribozyme, a hepatitis delta virus (HDV) ribozyme, a Csy4, and a tRNA-derived sequence. In some embodiments, the BPMV vector includes BPMV genomic segment RNA2. In the BPMV vector, the BPMV-RNA2 carries a guide RNA. In the BPMV vector, the BPMV-RNA2 is linked to a guide RNA. In some embodiments, the BPMV vector includes BPMV genomic segment RNA1. In the BPMV vector, the BPMV-RNA1 carries a guide RNA. In the BPMV vector, the BPMV-RNA1 is linked to a guide RNA. Cas nuclease(s) can be linked to part of the BPMV vector, and/or overexpressed in the plant. In some embodiments, the soybean plant overexpresses the Cas nuclease. The BPMV infects the soybean plant and delivers carried gene editing components to the meristem cells. The BPMV infects the soybean plant and delivers gene editing components (such as guide RNA) to the meristem cells. A guide RNA can be delivered to the meristem cells by means of BPMV viral delivery, such means including, but not limited to, direct leaf rub inoculation with infectious sap, inoculation with an infectious cDNA plasmid, and transformation of the root with a bacterium comprising a vector comprising the bean pod mottle virus (BPMV). A guide RNA can be delivered to the meristem cells by means of BPMV viral delivery, such means including, but not limited to, direct leaf rub inoculation with infectious lysate, inoculation with an infectious cDNA plasmid, and transformation of the root with a bacterium comprising a vector comprising the bean pod mottle virus (BPMV). As plant viruses are designed to deliver genetic cargo to plant cells, utilization of such methods to deliver genomic editing reagents is very effective. A soybean plant may be edited at a given locus by such methods without the need for integration of gene editing system components into the genome of the plants, such that the progeny of the plant inherits the edit but is free of gene editing components.

[0094] In some embodiments, the genomic target is in a scion. In some embodiments, the Cas nuclease is delivered to the scion by transport from a grafted rootstock. Accordingly, the present application also provides, in some embodiments, methods of editing a genomic target in a scion comprising grafting the scion onto a rootstock expressing a Cas nuclease, wherein the rootstock comprises nucleic acid encoding the Cas nuclease fused to a meristem transport segment (MTS); and delivering a guide RNA for the Cas nuclease to the scion by virus-mediated delivery, optionally wherein the scion comprises a leaf, a shoot, a stem, or other vegetative tissue. The rootstock provides a Cas nuclease to the scion, transported through the grafting site due to the MTS. Any guide RNA can then be delivered to the scion by means of viral delivery, such mean including but not limited to direct leaf rub inoculation with infectious sap, inoculation with an infectious cDNA plasmid, and transformation of the root with a bacterium comprising a vector comprising a recombinant plant virus. Any guide RNA can then be delivered to the scion by means of viral delivery, such mean including but not limited to direct leaf rub inoculation with infectious lysate, inoculation with an infectious cDNA plasmid, and transformation of the root with a bacterium comprising a vector comprising a recombinant plant virus As plant viruses are designed to deliver genetic cargo to plant cells, utilization of such methods to deliver genomic editing reagents is very effective. A scion may be edited at a given locus by such methods without the need for a transgene insertion in the genome of the scion. By other methods, integration of edits or desired traits into elite germplasm would take many crosses and require additional time and resources.

[0095] The present application provides methods of editing a genomic target in a scion comprising grafting the scion onto a rootstock expressing a Cas nuclease, wherein the rootstock comprises nucleic acid encoding the Cas nuclease fused to a meristem transport segment (MTS); and delivering a guide RNA for the Cas nuclease to the scion by virus-mediated delivery. The rootstock provides a Cas nuclease to the scion, transported through the grafting site due to the MTS. Any guide RNA can then be delivered to the scion by means of viral delivery, such mean including but not limited to direct leaf rub inoculation with infectious sap, inoculation with an infectious cDNA plasmid, and transformation of the root with a bacterium comprising a vector comprising a recombinant plant virus. Any guide RNA can then be delivered to the scion by means of viral delivery, such mean including but not limited to direct leaf rub inoculation with infectious lysate, inoculation with an infectious cDNA plasmid, and transformation of the root with a bacterium comprising a vector comprising a recombinant plant virus. As plant viruses are designed to deliver genetic cargo to plant cells, utilization of such methods to deliver genomic editing reagents is very effective. A scion may be edited at a given locus by such methods without the need for a transgene insertion in the genome of the scion. By other methods, integration of edits or desired traits into elite germplasm would take many crosses and require additional time and resources.

[0096] The present application provides methods of infecting a soybean plant that has previously been engineered to express a Cas nuclease, the method comprising the use of viral vectors. In some embodiments, the viral vector is a BPMV vector. In some embodiments, the viral vector comprises a guide RNA. This method of infecting a soybean plant is utilized to produce an edited soybean meristem cell. One advantage of this method is that the guide RNA does not carry over to the soybean plant’s progeny. Another advantage of this method is that engineering Cas nuclease-overexpressing soybean plants prior to infection can circumvent the challenge of limited BPMV cargo size, which would otherwise need to deliver the Cas nuclease as well.

A. Editing of a soybean plant by combined expression of Cas and Guide RNA

[0097] The present application provides methods of editing a genomic target in a meristem cell of a soybean plant, comprising delivering a guide RNA (gRNA) directed to the genomic target in the meristem cell in the soybean plant by virus-mediated delivery; and delivering a Cas nuclease to the meristem cell, wherein the Cas nuclease and the guide RNA edit the genomic target in the meristem cell of the soybean plant.

[0098] In some embodiments, the genome editing reagents are provided to the soybean plant by infection with a BPMV vector. In some embodiments, the BPMV vector carries at least one guide RNA. In some embodiments, the BPMV vector is linked to at least one guide RNA. In some embodiments, the Cas enzyme is delivered to the soybean plant in the same BPMV vector carrying the gRNA. In some embodiments, the Cas enzyme is delivered to the soybean plant by a second BPMV vector comprising nucleic acid encoding the Cas enzyme. In some embodiments, the method further comprises infecting the soybean plant with a plurality of vectors, each vector comprising a gRNA and/or the Cas enzyme. In some embodiments, the plurality of BPMV vectors co-infect the soybean plant simultaneously or infect the soybean plant in more than one round of infection. In some embodiments, the BPMV vector also includes nucleic acid encoding Casl2f nuclease. In some embodiments, nucleic acid encoding Casl2f nuclease and the guide RNA are provided in the same BPMV vector. In some embodiments, the nucleic acid encoding Casl2f nuclease and the guide RNA are provided in different BPMV vectors. In some embodiments, nucleic acid encoding the Cas nuclease and the guide RNA are provided in the same BPMV vector. In some embodiments, the nucleic acid encoding the Cas nuclease and the guide RNA are provided in different BPMV vectors. Also provided in the present disclosure is a viral vector system for use in soybean editing, the system comprising: a plant virus genome component; a guide RNA (gRNA) directed to a genomic target in soybean; and a nucleic acid encoding Casl2f nuclease. The methods and soybean plants of the present disclosure confer the advantage that no guide RNA remains in the progeny of the soybean plant. In some embodiments, no guide RNA remains in the gametes produced by the edited soybean meristem cells herein. In some embodiments, no guide RNA remains in the soybean seed generated by the methods herein.

[0099] In some embodiments, the soybean plant expresses a Cas nuclease prior to infection with the BPMV vector. In some embodiments, the soybean plant is a Cas editor line. In some embodiments, the soybean plant has a Cas enzyme stably integrated into its genome. In some embodiments, the genome editing reagents are overexpressed in the soybean plant. In some embodiments, the soybean plant overexpresses the Cas enzyme. In some embodiments, the soybean plant overexpresses Cas9. In some embodiments, the expression of the nuclease is under control of an inducible promoter. The soybean plant overexpressing a Cas nuclease may be generated through transformation techniques. In some embodiments, the soybean plant overexpresses Casl2, such as Casl2f. In some embodiments, the soybean plant is engineered to express a Cas nuclease prior to viral vector delivery. Infecting a soybean plant expressing a Cas nuclease may be advantageous, as this method avoids the need to deliver the Cas nuclease via highly limited viral vector cargo space. In some embodiments, the soybean plant overexpresses the Cas in a meristem cell. In some embodiments, the soybean plant overexpresses the Cas in cells other than the meristem cell and the Cas is transported to the meristem cell via a MTS.

[0100] The present application also provides methods of editing a genomic target in a plant scion comprising grafting the scion onto a rootstock comprising nucleic acid encoding a Cas nuclease and nucleic acid encoding a guide RNA for the Cas nuclease, wherein the nucleic acid encoding the guide RNA and the nucleic acid encoding the Cas nuclease are fused to a nucleic acid encoding a meristem transport segment (MTS). A rootstock provides nucleic acid encoding genome editing reagents, i.e., a Cas nuclease and a guide RNA for the Cas nuclease, to the plant vascular system. In some embodiments, RNA encoding the Cas9 nickase or Cas 12 nuclease and the guide RNA are transported from the rootstock to the scion by the plant vascular system. In some embodiments, RNA encoding the Cas9 nickase or Cas 12 nuclease and the guide RNA are transported from the rootstock to the scion through the phloem. In some embodiments, RNA encoding the Cas9 nickase or Casl2 nuclease is translated in the scion. In some embodiments, a meristem cell of the scion is edited. [0101] Provided herein is a rootstock comprising nucleic acid encoding a Cas9 nickase or Casl2 nuclease and nucleic acid encoding a guide RNA for the Cas9 nickase or Casl2 nuclease, wherein the nucleic acid encoding the guide RNA and the nucleic acid encoding the Cas9 nickase or Casl2 nuclease are fused to nucleic acid encoding a meristem transport segment (MTS). In some embodiments, the Cas nuclease is fused to a meristem transport segment (MTS).

[0102] In some embodiments, the genome editing reagents are provided to the rootstock by infection with Agrobacterium rhizogenes (also known as Rhizobium rhizogenes), producing a rootstock with transgenic hairy roots. In some embodiments, the Cas enzyme is provided to the soybean plant by infection with Agrobacterium rhizogenes (also known as Rhizobium rhizogenes), producing a soybean plant with transgenic hairy roots. In some embodiments, the nucleic acid encoding the Cas9 nickase or Cas 12 nuclease and the nucleic acid encoding the guide RNA are provided in the same vector. In some embodiments, the nucleic acid encoding the Cas9 nickase or Cas 12 nuclease and the nucleic acid encoding the guide RNA (gRNA) are provided in different vectors. In some embodiments, the vector is a viral vector. In some embodiments, virus-mediated delivery comprises using a viral vector comprising the gRNA. In some embodiments, the vector is a T-DNA vector. In some embodiments, the vector is a viral vector or a T-DNA vector.

[0103] In some embodiments, virus-mediated delivery comprises infecting the soybean plant with an inoculum comprising the viral vector. The fusion of the BPMV-RNA2 to the gRNA and/or the nucleic acid encoding the Cas nuclease results in the genome editing reagent(s) being transported to cells of the meristem of the soybean plant through viral infection and viral invasion. Modifications or edits made in the soybean plant meristem are heritable as the meristem nuclei will form the reproductive tissues of the plant, including the gametes. Modifications or edits made in the soybean plant meristem are heritable as the meristem cells will form the reproductive tissues of the plant, including the gametes.

[0104] In some embodiments, virus-mediated delivery comprises infecting the soybean plant with an inoculum comprising the viral vector. The fusion of the BPMV-RNA1 to the gRNA and/or the nucleic acid encoding the Cas nuclease results in the genome editing reagent(s) being transported to cells of the meristem of the soybean plant through viral infection and viral invasion. Modifications or edits made in the soybean plant meristem are heritable as the meristem nuclei will form the reproductive tissues of the plant, including the gametes. Modifications or edits made in the soybean plant meristem are heritable as the meristem cells will form the reproductive tissues of the plant, including the gametes.

B. Editing of a grafted scion mediated by root expression of Cas and Guide RNA

[0105] 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 Cas nuclease and nucleic acid encoding a guide RNA for the Cas nuclease, wherein the nucleic acid encoding the guide RNA and the nucleic acid encoding the Cas nuclease are fused to a nucleic acid encoding a meristem transport segment (MTS). A rootstock provides nucleic acid encoding genome editing reagents, i.e., a Cas nuclease and a guide RNA for the Cas nuclease, to the plant vascular system. In some embodiments, RNA encoding the Cas9 nickase or Cas 12 nuclease and the guide RNA are transported from the rootstock to the scion by the plant vascular system. In some embodiments, RNA encoding the Cas9 nickase or Cas 12 nuclease and the guide RNA are transported from the rootstock to the scion through the phloem. In some embodiments, RNA encoding the Cas9 nickase or Casl2 nuclease is translated in the scion. In some embodiments, a meristem of the scion is edited.

[0106] Provided herein is a rootstock comprising nucleic acid encoding a Cas9 nickase or Cas 12 nuclease and nucleic acid encoding a guide RNA for the Cas9 nickase or Cas 12 nuclease, wherein the nucleic acid encoding the guide RNA and the nucleic acid encoding the Cas9 nickase or Cas 12 nuclease are fused to nucleic acid encoding a meristem transport segment (MTS).

[0107] In some embodiments, the genome editing reagents are provided to the rootstock by infection with Agrobacterium rhizogenes (also known as Rhizobium rhizogenes), producing a rootstock with transgenic hairy roots. In some embodiments, the nucleic acid encoding the Cas9 nickase or Casl2 nuclease and the nucleic acid encoding the guide RNA are provided in the same vector. In some embodiments, the nucleic acid encoding the Cas9 nickase or Cas 12 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 T-DNA vector. In some embodiments, the vector is a viral vector or a T-DNA vector.

[0108] In some embodiments, the rootstock comprises nucleic acid encoding two or more, three or more, four or more, or five or more guide RNAs. In some embodiments, the nucleic acid encoding each of the two or more, three or more, four or more, or five or more guide RNAs is joined to an MTS.

[0109] In some embodiments, the rootstock further comprises nucleic acid encoding a detectable marker fused to a nucleic acid encoding an MTS.

[0110] In some embodiments, a scion is grafted onto the rootstock. The fusion of the meristem transport segment to nucleic acid encoding the genome editing reagents results in the genome editing reagents 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 genome editing reagents 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. Nucleic acid encoding the genome editing reagents are translated in the cytosol of cells of the scion meristem and imported into meristem cells, whereupon the genome of the meristem cell is edited. Edits made in the scion meristem cell are heritable as the meristem cell will form the reproductive tissues of the plant, including the gametes.

[0111] By this method, 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 be genetically edited without containing sequences encoding the Cas nuclease and the guide RNA in its genome. This will result in more consistent editing results, as there will be no element of randomness as to where a transgene will insert itself in the genome, or what levels of expression will result from each randomized insertion locus. 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 genome editing reagents from the scion genome. Additionally, the provided line of rootstocks comprising genome editing reagents can be a modular tool for editing a number of existing elite plant lines. A single rootstock line can be used to transform many grafted scions, without the need to transform each scion. 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.

C. Delivery of guide RNA to edit a soybean plant

[0112] 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 Cas nuclease, wherein the nucleic acid encoding a Cas nuclease is fused to a nucleic acid encoding a meristem transport segment (MTS), and delivering to the scion a guide RNA for the Cas nuclease. The present application also provides methods of editing a genomic target in a meristem cell of a soybean plant comprising delivering a guide RNA (gRNA) directed to the genomic target in the meristem cell in the soybean plant by virus-mediated delivery; and delivering a Cas nuclease to the meristem cell, wherein the Cas nuclease and the guide RNA edit the genomic target in the meristem cell of the soybean plant, thereby editing the genomic target in the meristem cell. In some embodiments, the Cas nuclease is delivered to the plant by transformation methods. In some embodiments, the Cas enzyme is delivered to the plant by infection with Agrobacterium. In some embodiments, the infection with Agrobacterium comprises infection with Agrobacterium rhizogenes (also known as Rhizobium rhizogenes), producing a plant with transgenic hairy roots. A rootstock provides nucleic acid encoding a Cas nuclease to the plant vascular system. In some embodiments, a scion is grafted onto the rootstock. In some embodiments, the virus-mediated delivery comprises using a viral vector comprising the gRNA.

[0113] In some embodiments, the Cas nuclease is fused to a meristem transport segment (MTS). The fusion of the meristem transport segment to nucleic acid encoding the Cas nuclease results in the nucleic acid encoding the Cas nuclease 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 Cas nuclease is translated in the cytosol of cells of the scion meristem and imported into meristem nuclei. In some embodiments, RNA encoding the Cas nuclease is translated in the meristem. In some embodiments, the Cas nuclease is delivered by transport from another part of the plant through the plant vascular system. [0114] In some embodiments, the method comprises delivering 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. In some embodiments, two or more guide RNAs are encoded by a single precursor RNA. In some embodiments, the two or more guide RNAs are each flanked by a direct repeat. In some embodiments, the viral vector carries at least two guide RNAs, at least three guide RNAs, at least four guide RNAs, at least five guide RNAs, at least six guide RNAs, at least seven guide RNAs, or at least eight guide RNAs. In some embodiments, the method comprises delivering a viral vector carrying two or more, three or more, four or more, or five or more guide RNAs. In some embodiments, the viral vector comprises at least two gRNAs, at least three gRNAs, at least four gRNAs, at least five gRNAs, at least six gRNAs, at least seven gRNAs, or at least eight gRNAs, optionally wherein each gRNA is directed to a different genomic target in the soybean plant or a same genomic target in the soybean plant. In some embodiments, the two or more, three or more, four or more, or five or more guide RNAs are each joined to the viral vector. In some embodiments, each guide RNA is directed to a different gene of interest in the soybean plant. In some embodiments, each guide RNA is directed to the same gene of interest. In some embodiments, the two or more guide RNAs are each flanked by a direct repeat. In some embodiments, the BPMV vector carries at least two guide RNAs, at least three guide RNAs, at least four guide RNAs, at least five guide RNAs, at least six guide RNAs, at least seven guide RNAs, or at least eight guide RNAs. In some embodiments, the method comprises delivering a BPMV vector carrying 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 the BPMV vector.

[0115] A guide RNA may be delivered to the meristem in a variety of ways. A guide RNA may be delivered to the meristem cell in a variety of ways. For example, in some embodiments, the guide RNA is delivered to the scion or directly to the meristem of the scion. In some embodiments, the guide RNA is delivered to the rootstock and transported into the scion. In some embodiments, the guide RNA is produced in vitro. In some embodiments, the guide RNA is methylated in vitro, such as by an RNA methylase, to promote mobility. In some embodiments, the guide RNA is fused to a meristem transport segment (MTS). Delivery of the guide RNA can occur through the following non-exhaustive list: through use of an RNA spray comprising the guide RNA and a simple surfactant (see, e.g., U.S. Pat. No. 9,121,022); by application of a composition comprising the guide RNA 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 RNA; by injection of a composition comprising the guide RNA into the stem; by infiltration of the leaf with a composition comprising the guide RNA; by direct uptake in the roots of a composition comprising the guide RNA; or by biolistic delivery to leaves or other tissue with circular DNA expressing the guide RNA. In some embodiments, delivery of the guide RNA comprises spraying a composition comprising the guide RNA onto the leaves, shoot, stem, and/or meristem. In some embodiments, the composition comprising the guide RNA comprises a surfactant. In some embodiments, the composition comprising the guide RNA comprises glass beads coated with the guide RNA. In some embodiments, delivery of the guide RNA comprises rubbing a composition comprising the guide RNA onto the leaves, shoot, stem, and/or meristem. In some embodiments, the virus-mediated delivery comprises direct leaf rub inoculation with infectious sap comprising the guide RNA. In some embodiments, the virus-mediated delivery comprises direct leaf rub inoculation with infectious lysate comprising the guide RNA. In some embodiments, delivery of the guide RNA comprises injecting a composition comprising the guide RNA into the stem. In some embodiments, delivery of the guide RNA comprises leaf infiltration of a composition comprising the guide RNA into the leaf. In some embodiments, the leaf infiltration comprises forced infiltration using a needle-less syringe or vacuum pump. In some embodiments, the composition comprising the guide RNA comprises a nuclease inhibitor. In some embodiments, the nuclease inhibitor comprises an RNase inhibitor. In some embodiments, delivery of the guide comprises biolis tic transformation of nucleic acid encoding the guide RNA into the leaf, shoot, shoot, stem, and/or meristem. In some embodiments, the viral vector is delivered to a leaf, shoot, stem, root, or other vegetative tissue. In some embodiments, the biolistic transformation comprises transformation of circular DNA encoding the guide RNA.

[0116] Delivery of a viral vector carrying the guide RNA can occur, for example, by application of a composition comprising the viral vector carrying the guide RNA onto a leaf after rubbing the leaf with 200 grit sandpaper with a dowel, by injection of a composition comprising the viral vector carrying the guide RNA into the stem, or by infiltration of the leaf with a composition comprising the viral vector carrying the guide RNA. In some embodiments, infecting the soybean plant comprises applying an inoculum comprising the viral vector carrying the gRNA. In some embodiments, infecting the soybean plant comprises inoculating the soybean plant’s leaves, shoot, stem, roots, or other vegetative tissue with a composition comprising the viral vector carrying the gRNA.

[0117] In some embodiments, virus-mediated delivery comprises infecting the soybean plant with an inoculum comprising the viral vector. In some embodiments, infecting the soybean plant comprises applying an inoculum comprising a viral vector carrying the gRNA. In some embodiments, infecting the soybean plant comprises inoculating the soybean plant’s leaves, shoot, stem, roots, or other vegetative tissue with a composition comprising the viral vector carrying the gRNA.

[0118] In some embodiments, the composition comprising the viral vector carrying the gRNA is infectious sap. In some embodiments, the inoculum is infectious sap. In some embodiments, the infectious sap is provided by inoculating a first host plant with an infectious cDNA plasmid and collecting infectious sap from the first host plant (see Li & Hataya Virology J. 2019, 16:18; Tran et al. J. Virol Methods 2014, 201: 57-64), the cDNA plasmid comprising the recombinant plant virus, the guide RNA, and/or the Cas enzyme. In some embodiments, the inoculum is infectious sap, wherein the infectious sap is provided by performing leaf infiltration of soybean or non-soybean leaves with bacteria comprising an infectious cDNA plasmid and collecting infectious sap from the soybean or non-soybean leaves, thereby providing the infectious sap. In some embodiments, the virus-mediated delivery further 1 comprises selecting from the set of first host plants a selected plant that is highly infected with intact viral cargo comprising the recombinant plant virus, the gRNA, and/or the Cas nuclease; raising the selected plant; and collecting the infectious sap from the selected plant, the method optionally comprising inoculating a second host plant or a set of second host plants with the infectious sap from the first selected plant. In some embodiments, the infectious sap is provided by inoculating a set of first host plants with at least one infectious cDNA plasmid and collecting infectious sap from the first host plant, the cDNA plasmid comprising the recombinant plant virus; the gRNA; and/or the Cas nuclease. In some embodiments, the method further comprises selecting the first host plant that is highly infected with intact viral cargo comprising the recombinant plant virus, the guide RNA, and/or the Cas enzyme; raising the selected plants; and collecting the infectious sap from the selected plant (see Mandal et al. Plant Dis. 2002, 9: 939-944; Mandal et al. J. Virol Meth. 2008, 149: 195-198; Laidlaw EPPO Bulletin 1987, 17:81-89; Sundaresha et al. Physiol Mol Biol Plants 2012, 18(4): 365-369; Mahas et al. Methods Mol Biol. 2019, 1917: 311-326; Mahmood et al. Viruses 2023, 15(2): 531). In some embodiments, the composition comprising the viral vector carrying the gRNA is infectious lysate. The term “lysate” is understood to include, but not be limited to, sap. In some embodiments, the inoculum is infectious lysate. In some embodiments, the infectious lysate is provided by inoculating a first host plant with an infectious cDNA plasmid and collecting infectious lysate from the first host plant (see Li & Hataya Virology J. 2019, 16:18; Tran et al. J. Virol Methods 2014, 201: 57-64), the cDNA plasmid comprising the recombinant plant virus, the guide RNA, and/or the Cas enzyme. In some embodiments, the inoculum is infectious lysate, wherein the infectious lysate is provided by performing leaf infiltration of soybean or non-soybean leaves with bacteria comprising an infectious cDNA plasmid and collecting infectious lysate from the soybean or non-soybean leaves, thereby providing the infectious lysate. In some embodiments, the virus-mediated delivery further comprises selecting from the set of first host plants a selected plant that is highly infected with intact viral cargo comprising the recombinant plant virus, the gRNA, and/or the Cas nuclease; raising the selected plant; and collecting the infectious lysate from the selected plant, the method optionally comprising inoculating a second host plant or a set of second host plants with the infectious lysate from the first selected plant. In some embodiments, the infectious lysate is provided by inoculating a set of first host plants with at least one infectious cDNA plasmid and collecting infectious lysate from the first host plant, the cDNA plasmid comprising the recombinant plant virus; the gRNA; and/or the Cas nuclease. In some embodiments, the method further comprises selecting the first host plant that is highly infected with intact viral cargo comprising the recombinant plant virus, the guide RNA, and/or the Cas enzyme; raising the selected plants; and collecting the infectious lysate from the selected plant. In some embodiments, the first host plant is not soybean. In some embodiments, the first host plant or selected plant is Nicotiana or soybean. In some embodiments, the first host plant is soybean. In some embodiments, the inoculum is produced by raising the selected plants and processing plant tissue. In some embodiments, the inoculum is produced by raising the selected plants and grinding selected leaves, fresh or lyophilized, using pestle and mortar in presence of a buffer. In some embodiments, the buffer is 10 mM sodium phosphate buffer, pH 7.0.

[0119] In some embodiments, the virus-mediated delivery further comprises selecting from the set of first host plants a selected plant that is highly infected with intact viral cargo comprising the recombinant plant virus, the gRNA, and/or the Cas nuclease; raising the selected plant; and collecting the infectious lysate from the selected plant, the method optionally comprising inoculating a second host plant or a set of second host plants with the infectious lysate from the first selected plant. In some embodiments, a third plant is inoculated with the infectious lysate from the second host plant. In some embodiments, a fourth plant is inoculated with the infectious lysate from the third plant. In some embodiments, a fifth plant is inoculated with the infectious lysate from the fourth plant. This process may continue iteratively.

[0120] In some embodiments, the provided method of editing a genomic target in a scion comprises grafting the scion onto a rootstock expressing a Cas nuclease, wherein the rootstock comprises nucleic acid encoding the Cas nuclease fused to a meristem transport segment (MTS); and delivering a guide RNA for the Cas nuclease to the scion by virus-mediated delivery. In some embodiments, the method further comprises transforming the rootstock with nucleic acid encoding the Cas nuclease prior to grafting. In some embodiments, the scion comprises a leaf, a shoot, a stem, and/or a meristem. In other aspects, provided herein are methods of editing a genomic target in the meristem cell of a plant comprising transforming the root of the plant with nucleic acid encoding a Cas nuclease; and delivering a guide RNA for the Cas nuclease to a leaf, a shoot, a stem, and/or meristem of the plant by virus- mediated delivery, wherein the nucleic acid encoding the Cas nuclease is fused to a meristem transport segment (MTS). In some embodiments, the guide RNA is fused to a meristem transport segment (MTS). In some embodiments, delivery of the guide RNA comprises inoculating the leaves, shoot, stem, and/or meristem with a composition comprising a recombinant plant virus that comprises the guide RNA or a polynucleotide encoding the guide RNA. In some embodiments, delivery of the guide RNA comprises transforming the plant with a bacterium comprising a binary vector comprising a recombinant plant virus. In some embodiments, delivery of the guide RNA comprises transforming the leaves, shoot, stem, and/or meristem of the plant with a bacterium comprising a binary vector comprising a recombinant plant virus. In some embodiments, infecting the soybean plant comprises inoculating the soybean plant’s leaves, shoot, stem, roots, or other vegetative tissue with a composition comprising the viral vector that contains the gRNA. In some embodiments, delivery of the guide RNA comprises transforming the root of the plant with a bacterium comprising a binary vector comprising a recombinant plant virus. In some embodiments, the bacterium further comprises a binary vector comprising the Cas nuclease. In some embodiments, the virus-mediated delivery of the methods disclosed herein comprises transforming the root of the plant with a bacterium comprising a binary vector comprising a recombinant plant virus comprising the guide RNA or a nucleic acid encoding the guide RNA, optionally wherein the bacterium further comprises a binary vector comprising a nucleic acid encoding the Cas nuclease. [0121] In some embodiments, the composition comprising the recombinant plant virus is infectious sap. In some embodiments, the infectious sap is provided by inoculating the leaves of a host plant with an infectious cDNA plasmid and collecting infectious sap from the host plant (see Li & Hataya Virology J. 2019, 16:18; Tran et al. J. Virol Methods 2014, 201: 57-64). In some embodiments, the method further comprises selecting one or more intermediate host plants that are infected with viruses carrying intact cargo comprising the guide RNA, raising the selected plants, and collecting the infectious sap from the selected plants (see Mandal et al. Plant Dis. 2002, 9: 939-944; Mandal et al. J. Virol Meth. 2008, 149: 195-198; Laidlaw EPPO Bulletin 1987, 17:81-89; Sundaresha et al. Physiol Mol Biol Plants 2012, 18(4): 365-369; Mahas et al. Methods Mol Biol. 2019, 1917: 311-326; Mahmood et al. Viruses 2023, 15(2): 531). In some embodiments, the composition comprising the recombinant plant virus is infectious lysate. In some embodiments, the infectious lysate is provided by inoculating the leaves of a host plant with an infectious cDNA plasmid and collecting infectious lysate from the host plant (see Li & Hataya Virology J. 2019, 16:18; Tran et al. J. Virol Methods 2014, 201: 57-64). In some embodiments, the method further comprises selecting one or more intermediate host plants that are infected with viruses carrying intact cargo comprising the guide RNA, raising the selected plants, and collecting the infectious lysate from the selected plants In some embodiments the virus is Foxtail Mosaic Virus (FoMV). In some embodiments, the plant is corn. In some embodiments, intermediate host plants that are highly infected with viruses are identified by detecting expression levels of viral coat protein, and/or detecting the presence of intact viral cargo. Several methods of detecting protein expression are known in the art, including but not limited to Western blots and EEISA assays (enzyme-linked immunosorbent assays). Several methods of detecting viral cargo are known in the art, including but not limited to PCR-based methods. In some embodiments, intermediate host plants that are highly infected with viruses are identified by measuring levels of viral coat protein-encoding mRNA in the intermediate host plants by RT-qPCR. In some embodiments, the virus-mediated delivery comprises selecting from the set of first host plants a selected plant that is highly infected with intact viral cargo comprising the recombinant plant virus, the gRNA, and/or the Cas nuclease. In some embodiments, the selected plant is identified by detecting expression levels of viral coat protein, and/or detecting the presence of intact viral cargo, wherein detecting the presence of intact viral cargo optionally comprises sequencing infectious cDNA in the first host plant; and/or measuring levels of viral coat proteinencoding mRNA in the first host plant by RT-qPCR. In some embodiments, assaying the presence of intact viral cargo comprises sequencing infectious cDNAs in the intermediate host plant to confirm that no spontaneous mutations have accumulated in the cargo to be delivered. In some embodiments, detecting the presence of intact viral cargo comprises sequencing infectious cDNAs in the first host plant to confirm that no spontaneous mutations have accumulated in the cargo to be delivered. In some embodiments, assaying intact viral cargo comprises sequencing infectious cDNAs in the intermediate host plant, comparing the obtained sequence to the sequence of the provided infectious cDNA plasmid, and determining that the intact viral cargo is substantially identical or completely identical to the corresponding sequence of the provided infectious cDNA plasmid. In some embodiments, the infectious sap is provided by performing leaf infiltration of tobacco leaves with bacteria comprising an infectious cDNA plasmid, and collecting infectious sap from the tobacco leaves. In some embodiments, the infectious lysate is provided by performing leaf infiltration of tobacco leaves with bacteria comprising an infectious cDNA plasmid, and collecting infectious lysate from the tobacco leaves. Leaf infiltration with Agrobacterium is also referred to herein as “agroinfiltration”. Agroinfiltration includes but is not limited to syringe-based agroinfiltration and vacuum-based agroinfiltration. In syringe-based agroinfiltration, a composition comprising Agrobacterium is placed in a needleless syringe, which is placed against the underside of a leaf. The composition is then injected into the airspace within the leaf. In vacuum-based agroinfiltration, leaf tissue, leaves, or whole plants are submerged in a composition comprising Agrobacterium, which is contained within a vacuum chamber. Vacuum is applied, forcing air out of intercellular spaces within the leaves. Releasing the vacuum results in introduction of the composition into the leaves. In some embodiments, the virus-mediated delivery comprises direct leaf rub inoculation with infectious sap comprising the guide RNA. In some embodiments, the virus- mediated delivery comprises direct leaf rub inoculation with infectious lysate comprising the guide RNA.

[0122] In some embodiments, RNA encoding the Cas nuclease and/or the guide RNA is transported by plant vascular system. In some embodiments, 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 transported to the meristem. In some embodiments, RNA encoding the Cas nuclease is translated in the meristem. In some embodiments, one or more meristematic cells is edited or modified.

[0123] In some embodiments, the guide RNA is transported to the meristem of the plant scion, or is provided to the meristem of the plant scion directly. The guide RNA is imported into the meristem nuclei. Upon import of both the Cas nuclease and the guide RNA for the Cas nuclease 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.

[0124] The provided methods allow for fast and modular editing of a multitude of soybean, or Glycine, plants, including elite lines, without the introduction of a transgene to the genome of the edited plant and/or edited 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 Cas nuclease, and different guide RNAs can be delivered to the different plant scions. Because there isn’t a different transgene being inserted into a different location in each plant scion, this allows for direct 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.

[0125] Additionally, the provided methods allow for a reduced number of required transformation events. The rootstock providing the Cas nuclease can be used with a wide variety of delivered guide RNAs, increasing the modularity of the editing system.

D. Delivery of guide RNA to edit a scion

[0126] 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 Cas nuclease, wherein the nucleic acid encoding a Cas nuclease is fused to a nucleic acid encoding a meristem transport segment (MTS), and delivering to the scion a guide RNA for the Cas nuclease. In some embodiments, the Cas nuclease is delivered to the plant by infection with Agrobacterium rhizogenes (also known as Rhizobium rhizogenes), producing a plant with transgenic hairy roots. A rootstock provides nucleic acid encoding a Cas nuclease to the plant vascular system. In some embodiments, a scion is grafted onto the rootstock. The fusion of the meristem transport segment to nucleic acid encoding the Cas nuclease results in the nucleic acid encoding the Cas nuclease 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 Cas nuclease is translated in the cytosol of cells of the scion meristem and imported into meristem nuclei.

[0127] In some embodiments, the method comprises delivering 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. In some embodiments, two or more guide RNAs are encoded by a single precursor RNA. In some embodiments, the two or more guide RNAs are each flanked by a direct repeat.

[0128] A guide RNA may be delivered to the meristem in a variety of ways. For example, in some embodiments, the guide RNA is delivered to the scion or directly to the meristem of the scion. In some embodiments, the guide RNA is delivered to the rootstock and transported into the scion. In some embodiments, the guide RNA is produced in vitro. In some embodiments, the guide RNA is methylated in vitro, such as by an RNA methylase, to promote mobility. In some embodiments, the guide RNA is fused to a meristem transport segment (MTS). Delivery of the guide RNA can occur through the following non-exhaustive list: through use of an RNA spray comprising the guide RNA and a simple surfactant (see, e.g., U.S. Pat. No. 9,121,022); by application of a composition comprising the guide RNA 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 RNA; by injection of a composition comprising the guide RNA into the stem; by infiltration of the leaf with a composition comprising the guide RNA; by direct uptake in the roots of a composition comprising the guide RNA; or by biolistic delivery to leaves or other tissue with circular DNA expressing the guide RNA. In some embodiments, delivery of the guide RNA comprises spraying a composition comprising the guide RNA onto the leaves, shoot, stem, and/or meristem. In some embodiments, the composition comprising the guide RNA comprises a surfactant. In some embodiments, the composition comprising the guide RNA comprises glass beads coated with the guide RNA. In some embodiments, delivery of the guide RNA comprises rubbing a composition comprising the guide RNA onto the leaves, shoot, stem, and/or meristem. In some embodiments, delivery of the guide RNA comprises injecting a composition comprising the guide RNA into the stem. In some embodiments, delivery of the guide RNA comprises leaf infiltration of a composition comprising the guide RNA into the leaf. In some embodiments, the leaf infiltration comprises forced infiltration using a needle-less syringe or vacuum pump. In some embodiments, the composition comprising the guide RNA comprises a nuclease inhibitor. In some embodiments, the nuclease inhibitor comprises an RNase inhibitor. In some embodiments, delivery of the guide comprises biolistic transformation of nucleic acid encoding the guide RNA into the leaf, shoot, shoot, stem, and/or meristem. In some embodiments, the biolistic transformation comprises transformation of circular DNA encoding the guide RNA.

[0129] In some embodiments, the provided method of editing a genomic target in a scion comprises grafting the scion onto a rootstock expressing a Cas nuclease, wherein the rootstock comprises nucleic acid encoding the Cas nuclease fused to a meristem transport segment (MTS); and delivering a guide RNA for the Cas nuclease to the scion by virus-mediated delivery. In some embodiments, the method further comprises transforming the rootstock with nucleic acid encoding the Cas nuclease prior to grafting. In some embodiments, the scion comprises a leaf, a shoot, a stem, and/or a meristem. In other aspects, provided herein are methods of editing a genomic target in the meristem of a plant comprising transforming the root of the plant with nucleic acid encoding a Cas nuclease; and delivering a guide RNA for the Cas nuclease to a leaf, a shoot, a stem, and/or meristem of the plant by virus-mediated delivery, wherein the nucleic acid encoding the Cas nuclease is fused to a meristem transport segment (MTS). In some embodiments, the guide RNA is fused to a meristem transport segment (MTS). In some embodiments, delivery of the guide RNA comprises inoculating the leaves, shoot, stem, and/or meristem with a composition comprising a recombinant plant virus that comprises the guide RNA or a polynucleotide encoding the guide RNA. In some embodiments, delivery of the guide RNA comprises transforming the plant with a bacterium comprising a binary vector comprising a recombinant plant virus. In some embodiments, delivery of the guide RNA comprises transforming the leaves, shoot, stem, and/or meristem of the plant with a bacterium comprising a binary vector comprising a recombinant plant virus. In some embodiments, delivery of the guide RNA comprises transforming the root of the plant with a bacterium comprising a binary vector comprising a recombinant plant virus. In some embodiments, the bacterium further comprises a binary vector comprising the Cas nuclease.

[0130] In some embodiments, the composition comprising the recombinant plant virus is infectious sap. In some embodiments, the infectious sap is provided by inoculating the leaves of a host plant with an infectious cDNA plasmid and collecting infectious sap from the host plant (see Li & Hataya Virology J. 2019, 16:18; Tran et al. J. Virol Methods 2014, 201: 57-64). In some embodiments, the method further comprises selecting one or more intermediate host plants that are infected with viruses carrying intact cargo comprising the guide RNA, raising the selected plants, and collecting the infectious sap from the selected plants (see Mandal et al. Plant Dis. 2002, 9: 939-944; Mandal et al. J. Virol Meth. 2008, 149: 195-198; Laidlaw EPPO Bulletin 1987, 17:81-89; Sundaresha et al. Physiol Mol Biol Plants 2012, 18(4): 365-369; Mahas et al. Methods Mol Biol. 2019, 1917: 311-326; Mahmood et al. Viruses 2023, 15(2): 531). In some embodiments, the composition comprising the recombinant plant virus is infectious lysate. In some embodiments, the infectious lysate is provided by inoculating the leaves of a host plant with an infectious cDNA plasmid and collecting infectious lysate from the host plant (see Li & Hataya Virology J. 2019, 16:18; Tran et al. J. Virol Methods 2014, 201: 57-64). In some embodiments, the method further comprises selecting one or more intermediate host plants that are infected with viruses carrying intact cargo comprising the guide RNA, raising the selected plants, and collecting the infectious lysate from the selected plants. In some embodiments the virus is Foxtail Mosaic Virus (FoMV). In some embodiments, the plant is corn. In some embodiments, intermediate host plants that are highly infected with viruses are identified by detecting expression levels of viral coat protein, and/or detecting the presence of intact viral cargo. Several methods of detecting protein expression are known in the art, including but not limited to Western blots and EEISA assays (enzyme- linked immunosorbent assays). Several methods of detecting viral cargo are known in the art, including but not limited to PCR-based methods. In some embodiments, intermediate host plants that are highly infected with viruses are identified by measuring levels of viral coat protein-encoding mRNA in the intermediate host plants by RT-qPCR. In some embodiments, assaying the presence of intact viral cargo comprises sequencing infectious cDNAs in the intermediate host plant to confirm that no spontaneous mutations have accumulated in the cargo to be delivered. In some embodiments, assaying intact viral cargo comprises sequencing infectious cDNAs in the intermediate host plant, comparing the obtained sequence to the sequence of the provided infectious cDNA plasmid, and determining that the intact viral cargo is substantially identical or completely identical to the corresponding sequence of the provided infectious cDNA plasmid. In some embodiments, the infectious sap is provided by performing leaf infiltration of tobacco leaves with bacteria comprising an infectious cDNA plasmid, and collecting infectious sap from the tobacco leaves. In some embodiments, the infectious lysate is provided by performing leaf infiltration of tobacco leaves with bacteria comprising an infectious cDNA plasmid, and collecting infectious lysate from the tobacco leaves. Eeaf infiltration with Agrobacterium is also referred to herein as “agroinfiltration”. Agroinfiltration includes but is not limited to syringe-based agroinfiltration and vacuum-based agroinfiltration. In syringe-based agroinfiltration, a composition comprising Agrobacterium is placed in a needleless syringe, which is placed against the underside of a leaf. The composition is then injected into the airspace within the leaf. In vacuum-based agroinfiltration, leaf tissue, leaves, or whole plants are submerged in a composition comprising Agrobacterium, which is contained within a vacuum chamber. Vacuum is applied, forcing air out of intercellular spaces within the leaves. Releasing the vacuum results in introduction of the composition into the leaves. In some embodiments, delivery of the guide RNA comprises direct leaf rub inoculation with infectious sap. In some embodiments, delivery of the guide RNA comprises direct leaf rub inoculation with infectious lysate.

[0131] In some embodiments, RNA encoding the Cas nuclease and/or the guide RNA is transported by plant vascular system. In some embodiments, 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 transported to the meristem. In some embodiments, RNA encoding the Cas nuclease is translated in the meristem. In some embodiments, one or more meristematic cells is edited.

[0132] The guide RNA is transported to the meristem of the plant scion, or is provided to the meristem of the plant scion directly. The guide RNA is imported into the meristem nuclei. Upon import of both the Cas nuclease and the guide RNA for the Cas nuclease 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.

[0133] 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 Cas nuclease, and different guide RNAs can be delivered to the different plant scions. Because there isn’t a different transgene being inserted into a different location in each plant scion, this allows for direct 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.

[0134] Additionally, the provided methods allow for a reduced number of required transformation events. The rootstock providing the Cas nuclease can be used with a wide variety of delivered guide RNAs, increasing the modularity of the editing system.

E. Uptake of guide RNA by roots for editing a plant

[0135] The present application provides methods of editing a genomic target in a plant meristem comprising providing a plant comprising nucleic acid encoding a Cas nuclease, wherein the nucleic acid encoding a Cas nuclease is fused to a nucleic acid encoding a meristem transport segment (MTS), and delivering to the root of the plant a guide RNA for the Cas nuclease. In some embodiments, the plant comprising the nucleic acid encoding a Cas nuclease is a rootstock. In some embodiments, a scion is grafted onto the rootstock. In some embodiments, the genomic editing reagents are provided to the plant by infection with Agrobacterium rhizogenes (also known as Rhizobium rhizogenes), producing a plant with transgenic hairy roots. In some embodiments, the Cas nuclease is delivered to the plant root by infection with Agrobacterium rhizogenes (also known as Rhizobium rhizogenes), producing a plant with transgenic hairy roots. The plant provides nucleic acid encoding a Cas nuclease to the plant vascular system. The fusion of the meristem transport segment to nucleic acid encoding the Cas nuclease results in the nucleic acid encoding the Cas nuclease being transported to cells of the meristem of the scion through the plant vascular system. In some embodiments, the nucleic acid encoding the Cas nuclease is transported from the rootstock to the scion through the graft junction. In some embodiments, RNA encoding the Cas nuclease and/or the guide RNA is transported by plant vascular system. In some embodiments, RNA encoding the Cas nuclease and/or the guide RNA is transported through the xylem or the phloem. 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. Nucleic acid encoding the Cas nuclease is translated in the cytosol of cells of the scion meristem and imported into meristem nuclei.

[0136] In some embodiments, the guide RNA is delivered to the roots. In some embodiments, the guide RNA is delivered via direct uptake in the roots. In some embodiments, the guide RNA is delivered to the plant by infection with Agrobacterium rhizogenes (also known as Rhizobium rhizogenes), producing a plant with transgenic hairy roots. In some embodiments, the guide RNA is injected into the roots. In some embodiments, the guide RNA is produced in vitro. In some embodiments, the guide RNA is methylated in vitro, such as by an RNA methylase, to promote mobility. In some embodiments, the guide RNA is fused to a meristem transport segment (MTS). Delivery of the guide RNA can occur through the following non-exhaustive list: through use of an RNA spray comprising the guide RNA and a simple surfactant (see, e.g., U.S. Pat. No. 9,121,022); by injection of a composition comprising the guide RNA into the stem; by direct uptake in the roots of a composition comprising the guide RNA; or by biolistic transformation of roots or other tissue with circular DNA expressing the guide RNA. The guide RNA is transported to the meristem of the plant, and is imported into the meristem nuclei. Upon import of both the Cas nuclease and the guide RNA for the Cas nuclease into the meristem nuclei, the genome of the meristem nuclei is edited. In some embodiments, upon import of the guide RNA for the Cas nuclease into the meristem nuclei where the Cas nuclease is expressed, 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 guide RNA is transported to the meristem of the soybean plant, and is imported into the meristem cell. Upon import of both the Cas nuclease and the guide RNA for the Cas nuclease into the meristem cell, the genome of the meristem cell is edited. Edits made in the scion meristem cell are heritable as the meristem cell will form the reproductive tissues of the plant, including the gametes. [0137] 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 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.

[0138] The provided methods for editing a plant transformed with Agrobacterium rhizogenes allow for a fast and modular introduction of heritable edits. A strain of Agrobacterium is developed that comprises the Cas nuclease, 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 RNA 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 an edited genomic target, and is thus an improvement on current editing techniques. 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.

F. Grafting

[0139] In some embodiments, the method provided herein comprise editing a grafted scion. The present disclosure utilizes grafting systems and their vascular mobility in some embodiments to accomplish VIGE. 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.

[0140] A successful graft exhibits a continuous vascular system from rootstock to scion, including transmission through a graft junction. RNAs and/or endonucleases expressed in the rootstock, in some embodiments encoding genome editing reagents, enter the phloem and transit to the shoot apical meristem of the scion. The RNAs and/or endonucleases are imported into cells of the meristem and are processed into functional RNPs, which are able to modify 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 genome editing reagents into an easily transformable germplasm that can then be grafted to elite shoots as a rootstock, resulting in heritable genome edits in the scion. [0141] A plant scion transformed through the present methods of genomic editing does not contain transgenes encoding the reagents for genomic modification. 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.

G. Configurations of viral vector systems

[0142] The recombinant plant virus systems, viral vector systems, and BPMV vector systems of the present disclosure may be modified as desired, including with processing elements, in order to optimize efficiency of the infection and promote excision of the guide RNA from a viral transcript.

[0143] Guide RNA(s) can be part of the same RNA (mRNA) capable of expressing a viral protein, such as RNA1 or RNA2. Guide RNAs can also be part of the same mRNA as the RNA encoding the Cas nuclease. Guide RNAs can also be provide in arrays comprising multiple guide RNAs with different sequences. In one embodiment, one or more guide RNAs are flanked by direct repeats (DR). In some embodiments, guide RNAs are excised by a Cas nuclease. In some embodiments, the two or more guide RNAs are each flanked by a direct repeat. For example, a translated and expressed active Casl2 nuclease can process Casl2 DR-flanked spacers of the mRNA to make guide RNAs. In alternative embodiments, 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). Among exemplary processing elements, “tRNAs” includes tRNA-derived sequences. 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, RNase H, 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.

[0144] In some embodiments, a viral vector is used for editing a genomic target in a meristem cell of a soybean plant. In some embodiments, the viral vector comprises a BPMV vector. In some embodiments, the viral vector is modified to improve or optimize editing efficiency. In some embodiments, the viral vector comprises: a first direct repeat, a spacer sequence complementary to the gene of interest, and a second direct repeat. In some embodiments, the nucleic acid encoding the guide RNA and/or the Cas enzyme is located between two ribozyme sequences. In some embodiments, 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-derived sequence sequence. In some embodiments, the guide RNA is processed by a ribozyme selected from the group consisting of a hammerhead ribozyme, a hepatitis delta virus (HDV) ribozyme, a Csy4, and a tRNA- derived sequence. In some embodiments, the viral vector comprises: a first ribozyme sequence, a direct repeat, a spacer sequence complementary to the gene of interest, and a second ribozyme sequence, wherein the first ribozyme sequence and the second ribozyme sequence are the same or different sequences. In some embodiments, the viral vector carrying the guide RNA or a nucleic acid encoding the guide RNA further comprises a hammerhead ribozyme sequence 5’ to the nucleic acid encoding the guide RNA, and a HDV ribozyme 3’ to the nucleic acid encoding the guide RNA. In some embodiments, the viral vector comprises: a RNase-resistant caged truncated pre-tRNA-like crRNA (catRNA), a first direct repeat, a spacer sequence complementary to the gene of interest, and a second direct repeat. In some embodiments, the BPMV vector further comprises the Cas enzyme. The Cas enzyme may be placed in any order relative to the guide RNA and other processing elements, as long as it is flanked by processing elements. In some embodiments, a guide RNA is encoded by DNA. In some embodiments, the guide RNA is fused to a bean pod mottle virus (BPMV) vector. Also provided by the present disclosure is a viral vector system for producing an edited genomic target in a soybean plant, the system comprising: a plant virus genome component; a guide RNA (gRNA) directed to the genomic target; and a Cas nuclease expressed in the meristem cell of the soybean plant. In some embodiments, the viral vector is a cDNA clone. In some embodiments, cDNA/DNA constructs comprising the viral vector are used in Agrobacterium transformation. In some embodiments, cDNA/DNA constructs comprising the viral vector are used in biolistic delivery. In some embodiment, the viral vector is delivered via plasmid. In some embodiments, the viral vector is an RNA vector. In some embodiments, the viral vector is delivered as RNA in the form of abrasive mechanical inoculation. [0145] Also provided in some aspects of the present disclosure is a method for making a heritable genomic modification at a target site in a soybean plant, the method comprising delivering a BPMV vector comprising the sequence of BPMV-RNA2 linked to a nucleic acid sequence encoding a guide RNA (gRNA) to a cell of the soybean plant, wherein the BPMV infects a meristem cell of the plant expressing a heterologous Cas nuclease and the gRNA is expressed in the same meristem; and allowing the gRNA and the Cas nuclease to form a complex and introduce a single- or double-strand break at the target site in the genome of the meristem’s cell or cells, thereby making a heritable genomic modification. Also provided in some aspects of the present disclosure is a method for making a heritable genomic modification at a target site in a soybean plant, the method comprising delivering a BPMV vector comprising the sequence of BPMV-RNA1 linked to a nucleic acid sequence encoding a guide RNA (gRNA) to a cell of the soybean plant, wherein the BPMV infects a meristem cell of the plant expressing a heterologous Cas nuclease and the gRNA is expressed in the same meristem; and allowing the gRNA and the Cas nuclease to form a complex and introduce a single- or double-strand break at the target site in the genome of the meristem’s cell or cells, thereby making a heritable genomic modification. In some embodiments, the BPMV vector comprises: a first ribozyme sequence, a direct repeat, a spacer sequence directed to the target site, and a second ribozyme sequence, wherein the first ribozyme sequence and the second ribozyme sequence are the same or different sequences. In some embodiments, the BPMV vector comprises: a first direct repeat, a spacer sequence directed to the target site, and a second direct repeat. In some embodiments, the BPMV vector comprises: a catRNA, a first direct repeat, a spacer sequence directed to the target site, and a second repeat. In some embodiments, the guide RNA is processed by a ribozyme selected from the group consisting of a hammerhead ribozyme, a hepatitis delta virus (HDV) ribozyme, a tRNA-derived sequence, or other potential processors such as Csy4.

[0146] The present disclosure provides a viral vector system for use in soybean editing, the system comprising: a plant virus genome component; one or more gRNA; and a direct repeat and/or selfcleaving ribozyme sequence flanking the one or more gRNA. Also provided is a bean pod mottle virus (BPMV) viral vector system comprising: a BPMV genome component; one or more gRNA; and a direct repeat and/or self-cleaving ribozyme sequence flanking the one or more gRNA.

H. CRISPR-Cas systems

[0147] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas systems, or CRISPR systems, are adaptive defense systems originally discovered in bacteria and archaea. CRISPR systems use RNA-guided nucleases termed CRISPR-associated or “Cas” endonucleases (e.g., Cas9 or Casl2a (“Cpfl”)) to cleave foreign DNA. In a typical CRISPR/Cas system, 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” that target single- or double-stranded DNA sequences. In microbial hosts, CRISPR loci encode both Cas endonucleases and “CRISPR arrays” of the non-coding RNA elements that determine the specificity of the CRISPR-mediated nucleic acid cleavage.

[0148] 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. 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). Some endonucleases, e.g., 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. Cas 12a (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 Cas 12a PAM sequences include those for the naturally occurring Acidaminococcus sp. BV3L6 Cpfl (AsCpfl) and Lachnospiraceae bacterium ND2006 Cpfl (LbCpfl) TTTV, where V can be A, C, or G. In some instances, 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 are known in the art and are routine, 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.

I. Nucleases

[0149] 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 Casl2a (“Cpfl”) CRISPR system includes the type V endonuclease Casl2a (also known as “Cpfl”). Casl2a nucleases are characterized as having only a RuvC nuclease domain, in contrast to Cas9 nucleases which have both RuvC and HNH nuclease domains. Casl2a 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. Examples of Casl2a nucleases include AsCasl2a or “AsCpfl” (from Acidaminococcus sp.) and LbCasl2a or “LbCpfl” (from Lachnospiraceae bacteria). In contrast to Cas9 type CRISPR systems, 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. Casl2f is another type of nuclease in the Casl2 family (alongside Casl2a, Casl2b, etc.), and its compact size makes it an excellent candidate for being included in a viral vector system.

[0150] It is understood that for all systems, the use of a 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) or nCas (“nickase” Cas, i.e., Cas that makes single-stranded breaks rather than double-stranded breaks), TALE (TAL-effector), or ZF (zinc finger) versions of the polypeptides. 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.

[0151] Useful CRISPR-based RNA-guided nuclease systems have been described and are known from the literature, including but not limited to Cas9, Casl2a (Cpfl), Casl2e (CasX), Casl2d (CasY), C2cl, C2c2, C2c3 (see WO2018176009), Casl2h, Casl2i (see Yan et al. Science 2019, 363(6422): 88- 91) and Casl2j (Pausch et al. Science 2020, 369(6501): 333-337). Useful RNA-guided nuclease systems have been described and are known from the literature, including but not limited to Cas9, Casl2a (Cpfl), Casl2e (CasX), Casl2d (CasY), C2cl, C2c2, C2c3 (see WO2018176009), Casl2h, Casl2i (see Yan et al. Science 2019, 363(6422): 88-91) and Casl2j (Pausch et al. Science 2020, 369(6501): 333- 337). “Casl2” is used herein to refer to any Casl2 protein, including but not limited to Casl2f, Casl2a (Cpfl), Casl2e (CasX), Casl2d (CasY), C2cl, C2c2, C2c3 (see WO2018176009), Casl2h, Casl2i (see Yan et al. Science 2019, 363(6422): 88-91) and Casl2j (Pausch et al. Science 2020, 369(6501): 333- 337. In some embodiments, the Cas nuclease is a nuclease selected from the group consisting of Cas9, Casl2f, Casl2a (Cpfl), Casl2e (CasX), Casl2d (CasY), C2cl, C2c2, C2c3, Casl2h, Casl2i, and Casl2j. In some embodiments, the Cas nuclease is a nuclease selected from the group consisting of Cas9, Casl2f, Casl2a (Cpfl), any Mini Cas, Casl2e (CasX), Casl2d (CasY), C2cl, C2c2, C2c3, Casl2h, Casl2i, and Casl2j, TnpB, IscB, and any omegaRNA. In some embodiments, the Cas nuclease is a Cas nickase. In some embodiments, the Cas nuclease is a Cas9 nickase or a Casl2 nuclease. In some embodiments, the Cas nuclease is a Cas9 nuclease or a Casl2 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.

[0152] It is understood that for all systems, the use of a 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) or nCas (“nickase” Cas, i.e., Cas that makes single-stranded breaks rather than double-stranded breaks), TALE (TAL-effector), or ZF (zinc finger) versions of the polypeptides. 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.

[0153] Useful CRISPR-based RNA-guided nuclease systems have been described and are known from the literature, including but not limited to Cas9, Casl2a (Cpfl), Casl2e (CasX), Casl2d (CasY), C2cl, C2c2, C2c3 (see WO2018176009), Casl2h, Casl2i (see Yan et al. Science 2019, 363(6422): 88- 91) and Casl2j (Pausch et al. Science 2020, 369(6501): 333-337). “Casl2” is used herein to refer to any Casl2 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. In some embodiments, the Cas nuclease is selected from the group consisting of Cas9, Casl2a (Cpfl), Casl2e (CasX), Casl2d (CasY), C2cl, C2c2, C2c3, Casl2h, Casl2i, and Casl2j. In some embodiments, the Cas nuclease is selected from the group consisting of Cas9, Casl2a (Cpfl), a Mini Cas, Casl2e (CasX), Casl2d (CasY), C2cl, C2c2, C2c3, Casl2h, Casl2i, and Casl2j. In some embodiments, the Cas nuclease is a Cas nickase. In some embodiments, the Cas nuclease is a Cas9 nickase or a Casl2 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.

[0154] In some embodiments, the RNA-guided nuclease of the present disclosure is an evolutionary progenitor of Cas endonuclease(s). In some embodiments, the RNA-guided nuclease is a member of the OMEGA system (Obligate Mobile Element Guided Activity system). In some embodiments, the RNA-guided nuclease is a TnpB nuclease and/or an IscB nuclease.

[0155] In embodiments of the present disclosure, the term “Cas enzyme” includes all RNA-guided nucleases.

[0156] In some embodiments, the gRNA and the Cas nuclease form a complex and introduce a single - or double-stranded break in the sequence of the genomic target. In some embodiments, the viral vector comprising the gRNA further comprises a Casl2f nuclease. In some embodiments, the viral vector comprising the gRNA does not comprise the Cas nuclease. In some embodiments, the viral vector comprising the Cas nuclease does not comprise the gRNA. In some embodiments, the viral vector comprising the gRNA further comprises a Mini Cas.

[0157] In a phenomenon termed “codon bias”, different organisms use specific codons more often than synonymous codons to encode for the same amino acid. Furthermore, efficiency of mRNA translation can be correlated with the use of the preferred codons over less frequently used codons. A nucleic acid can therefore be optimized for expression in a desired host by replacing codons less frequently used in that host with those more frequently used in the host. Codon bias varies across species, as well as across wider phylogenetic distance. Codon usage tables are known in the art (see, e.g., the “Codon Usage Database” at www[dot]kazusa[dot]or[dot]jp[forward slash]codon) and these tables can be adapted in a number of ways, as shown in Nakamura et al. (Nucl Acids Res 2000, 28: 292). Computer algorithms may also be used for codon optimization of a particular sequence for expression in a desired host, such as Gene Forge (Aptagen; Jacobus, PA). For use in plants, see e.g., Campbell and Gowri (Plant Physiol 1990, 92: 1-11) and Murray et al. (Nucl Acids Res 1989, 17: 477- 498).

[0158] A Cas nuclease is encoded by a nucleic acid. In one embodiment, the nucleic acid encoding the Cas nuclease is codon-optimized for use in a species of plant. In some embodiments, the Cas nuclease is codon-optimized for expression in dicots. In some embodiments, the Cas nuclease is codon- optimized for expression in soybean. In some embodiments, the Cas nuclease is codon-optimized for expression in monocots. In some embodiments, the Cas nuclease is codon-optimized for expression in corn. In some embodiments, the Cas nuclease is codon-optimized for expression in wheat. In some embodiments, the Cas nuclease is fused to a nuclear localization signal (NLS). 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.

[0159] The nucleic acid encoding the Cas nuclease is fused to a nucleic acid encoding a meristem transport segment (MTS). In some embodiments, 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. In some embodiments, RNA encoding the Cas nuclease and at least one guide RNA are transported from the rootstock to the scion by the plant vascular system. In some embodiments, 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. In some embodiments, RNA encoding the Cas nuclease and at least one guide RNA are transported from the scion to the rootstock through the phloem. In some embodiments, 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 cell is on the plant scion.

[0160] In some embodiments, the nucleic acid encoding the Cas enzyme is linked to a promoter. For use in plants, 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). In some embodiments, the nucleic acid encoding the Cas enzyme is linked to a constitutive promoter. Examples of 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. 7,151,204, an opaline synthase (NOS) and octopine synthase (OCS) promoter from Agrobacterium tumefaciens, and a ubiquitin promoter. In some embodiments, the nucleic acid encoding the Cas enzyme is 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. 6,140,078 (salt inducible promoters), U.S. Pat. No. 6,252,138 (pathogen inducible promoters), and U.S. Pat. No. 6,175,060 (phosphorus deficiency inducible promoters). In some embodiments, the nucleic acid encoding the Cas enzyme is linked to a promoter selected from the group consisting of promoters active in roots and promoter active in phloem companion cells.

[0161] In some embodiments, the promoter is a constitutive promoter, optionally wherein the constitutive promoter is a ubiquitin promoter. In some embodiments, the promoter is selected from the group consisting of a promoter from a Arabidopsis WRKY6 gene, a promoter from a chickpea WRKY31 gene, a promoter from a carrot MYB113 gene, a promoter from a corn GLU1 gene, a promoter from a strawberry RB7-type TIP-2 gene, a promoter from a banana TIP2-2 gene, 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 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, or the promoter of an orthologous gene thereof. In some embodiments, the promoter is active in roots and/or phloem companion cells. In some embodiments, 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.

[0162] In some embodiments, 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). In some embodiments, 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. Plant J 1997, 12(5): 1179-1188), an RmlC-like cupins superfamily protein promoter (CN102002498B), a Commelina yellow mottle virus promoter (Medberry et al., Plant Cell 1992, 4: 185-192), a wheat dwarf virus promoter (W02003060135A2), a sucrose synthase promoter (Yang and Russell PNAS 1990, 87: 4144-4148), a glutamine synthetase promoter (Edwards et al. PNAS 1990, 87: 3459-3463), a phloem-specific isoform of plasmamembrane H+-ATPase promoter (DeWitt et al. Plant J. 1991, 1(1): 121-128), a JmjC domain-containing protein 18 (JMJ18) promoter (Yang et al., PLoS Genet 2012, 8(4): el002664), and a phloem protein 2 (PP2) promoter (US5495007A).

[0163] In some embodiments, 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, optionally 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. In some embodiments, the TLS comprises the nucleotide sequence set forth in SEQ ID NO: 29 or 30.

[0164] The nucleic acid encoding the Cas nuclease and/or at least one guide RNA may be transcribed in a rootstock. In some embodiments, the nucleic acid encoding the Cas nuclease is fused to a meristem transport segment (MTS). In some embodiments, the nucleic acid encoding the MTS is located 3’ of the nucleic acid encoding the Cas nuclease. In some embodiments, the nucleic acid encoding the MTS is located 5’ of the nucleic acid encoding the Cas nuclease. In some embodiments, the nucleic acid encoding the MTS is located 3’ of the nucleic acid encoding the Cas nuclease. In some embodiments, the nucleic acid encoding the MTS is located both 5’ and 3’ of the nucleic acid encoding the Cas nuclease. In some embodiments, 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. In some embodiments, RNA encoding the Cas nuclease and/or the guide RNA is transported by plant vascular system. In some embodiments, RNA encoding the Cas nuclease and/or the guide RNA is transported from the rootstock to the scion by plant vascular system. In some embodiments, RNA encoding the Cas nuclease and/or the guide RNA is transported through the xylem or the phloem. In some embodiments, RNA encoding the Cas nuclease is translated in the scion. In some embodiments, RNA encoding the Cas nuclease 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 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). For example, 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).

[0165] The nucleic acid encoding the Cas nuclease and/or at least one guide RNA is intended to be transcribed in the rootstock. In some embodiments, the nucleic acid encoding the Cas nuclease is fused to a meristem transport segment (MTS). In some embodiments, the nucleic acid encoding the MTS is located 3’ of the nucleic acid encoding the Cas nuclease. In some embodiments, the nucleic acid encoding the MTS is located 5’ of the nucleic acid encoding the Cas nuclease. In some embodiments, the nucleic acid encoding the MTS is located 3’ 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. In some embodiments, RNA encoding the Cas nuclease and/or the guide RNA is transported by plant vascular system. In some embodiments, RNA encoding the Cas nuclease and/or the guide RNA is transported from the rootstock to the scion by plant vascular system. In some embodiments, RNA encoding the Cas nuclease and/or the guide RNA is transported through the xylem or the phloem. In some embodiments, 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. In some embodiments, RNA encoding the Cas nuclease and/or the guide RNA is transported to the meristem cell, wherein the Cas nuclease and/or the guide RNA is translated in the meristem cell. 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). For example, 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).

[0166] The nucleic acid encoding the Cas nuclease can be optimized to increase nuclease activity and editing efficiency. In some embodiments, the nucleic acid encoding the Cas enzyme is linked to a nuclear localization signal (NLS), such as the NLS from SV40. Various NLSs, including those that bind to the major groove and/or the minor groove of an importin protein, are well known in the art, as in Kosugi et al. (J Biol Chem 2009, 284(1): 478-485). In some embodiments, 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). In some embodiments, the nucleic acid encoding the Cas enzyme further comprises a terminator. By “terminator” 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. Such 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. Gene, a 3' element from a non-human animal gene, a 3' element from a human gene, and a 3' element from a plant gene, wherein the 3' element terminate transcription of an RNA transcript located immediately 5' to the 3' element. Useful 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. 6,090,627, incorporated herein by reference; 3' elements from plant genes such as the heat shock protein 17, ubiquitin, and fructose- 1 ,6-biphosphatase genes from wheat (Triticum aestivum), and the glutelin, lactate dehydrogenase, and beta-tubulin genes from rice (Oryza saliva), disclosed in U.S. Patent Application Publication 2002/0192813 Al, incorporated herein by reference; in some embodiments, 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). In some embodiments, the nucleic acid encoding the Cas enzyme further comprises one or more introns. In some embodiments, the nucleic acid encoding the Cas enzyme further comprises one or more transcriptional enhancers. In some embodiments, the one or more transcriptional enhancers comprise one or more bacterial octopine synthase (OCS) enhancers (U.S. Patent No. 11,198,885). In one embodiment, the nucleic acid encoding the Cas enzyme further comprises a triple OCS enhancer (U.S. Patent No. 11,198,885). In some embodiments, the nucleic acid encoding the Cas enzyme further comprises a 5’ UTR comprising a translational enhancer. In some embodiments, the nucleic acid encoding the Cas enzyme further comprises a Kozak sequence endogenous to the scion species at the translation start codon. In some embodiments, the nucleic acid encoding the Cas enzyme further comprises nuclear localization signals flanking the coding sequence of the Cas enzyme.

[0167] In some embodiments, a viral vector or viral vector system is delivered to a soybean plant that already overexpresses a Cas nuclease. In some embodiments, the soybean plant overexpresses Cas9. This overexpression of a Cas nuclease may be the result of gene editing conducted before the viral infection of the soybean plant, and this overexpression can be produced through a variety of gene editing methods known in the art, not limited to viral infection of a viral vector carrying the Cas nuclease.

J. Guide RNAs

[0168] 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 “transactivating 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. In contrast, Cas 12a type CRISPR systems have been reported to function without a tracrRNA, with the Casl2a CRISPR arrays processed into mature crRNAs without the requirement of a tracrRNA; see Zetsche et al. Cell 2015, 163: 759-771 and U.S. Pat. No. 9,790,490. 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).

[0169] As used herein “guide RNA” or “gRNA” 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. In embodiments, a gRNA is a CRISPR RNA (“crRNA”), such as the engineered Casl2a crRNAs described in this disclosure. For nucleases (such as a Cas9 nuclease) that require a combination of a trans-activating crRNA (“tracrRNA”) and a crRNA for the nuclease to cleave the target nucleotide sequence, 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. 9,790,490, which is incorporated herein by reference in its entirety and particularly for its disclosure of DNA encoding Casl2a (Cpfl) endonucleases and guide RNAs and PAM sites. In practice, 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. The design of effective guide RNAs for use in plant genome editing is disclosed in U.S. Patent Application Publication 2015/0082478 Al, the entire specification of which is incorporated herein by reference. Chemically modified sgRNAs have been demonstrated to be effective in Cas9 genome editing; see, for example, Hendel et al. Nature Biotechnol., 2015, 33:985-991.

[0170] Guide RNA(s) can be part of the same RNA (mRNA) capable of expressing the Cas nuclease. In one embodiment, one or more guide RNAs are flanked by direct repeats (DR) of the CRISPR array from which the Cas effector polypeptide was first isolated. In some embodiments, the two or more guide RNAs are each flanked by a direct repeat. For example, a translated and expressed active Casl2a nuclease can process the DR-flanked spacers of the mRNA to make guide RNAs. In certain embodiments, a translated and expressed active Casl2a nuclease can process Casl2a DR-flanked spacers of the mRNA to make guide RNAs. In certain embodiments, a translated and expressed active Casl2e nuclease can process Casl2e DR-flanked spacers of the mRNA to make guide RNAs. In certain embodiments, a translated and expressed active Casl2i nuclease can process Casl2i DR-flanked spacers of the mRNA to make guide RNAs. In certain embodiments, a translated and expressed active Casl2j nuclease can process Casl2j DR-flanked spacers of the mRNA to make guide RNAs. In alternative embodiments, 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. In some embodiments, the nucleic acid encoding the guide RNA and the MTS is located between two ribozyme sequences. In some embodiments, 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-derived sequence. In some embodiments, 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. In some embodiments, a guide RNA is encoded by a nucleic acid. In some embodiments, the guide RNA is fused to a meristem transport segment (MTS). In some embodiments, 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 Cast 2 nuclease and/or 5’ of the nucleic acid encoding the guide RNA. In some embodiments, the nucleic acid encoding the MTS is located 3’ of the nucleic acid encoding the Cas9 nickase or Cast 2 nuclease and/or 3’ of the nucleic acid encoding the guide RNA. In some embodiments, the soybean plant further comprises a nucleic acid encoding a detectable marker fused to a nucleic acid encoding the MTS, optionally wherein the nucleic acid encoding the MTS is located 3’ or 5’ of a nucleic acid encoding the Cas nuclease.

[0171] In some embodiments, the guide RNA comprises at its 3’ end a priming site and an edit to be incorporated into the genomic target. In some embodiments, the nucleic acid encoding the guide RNA and the MTS further comprises a terminator. In some embodiments, the terminator is a U6 terminator. [0172] In some embodiments, the guide RNA comprises a 5 -methylcytosine group.

[0173] In some embodiments, 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. Either independently or additionally, 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. Either independently or additionally, a guide RNA which comprises labeled isotopes, such as one or more of ¹⁵N, ¹³C, ¹⁴C, Deuterium, or ³²P, or other atoms used as tracers, is a modified guide RNA. Either independently or additionally, 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’-0-methyl, 2’-deoxy, 2’ -methoxyethyl, 2’fluoro, 2’- amino, a sugar in L form, and 4’ -thioribosyl.

[0174] In certain embodiments, 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. In certain embodiments, 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. In some embodiments, the maintained functionality of the guide RNA includes binding both a Cas protein and a polynucleotide in complex. In some embodiments, the chemical modifications on the guide RNA are used to distinguish the sequences from the nascent sequences present in the experimental plant. In certain embodiments, 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.

[0175] In some embodiments, the guide RNA and the Cas nuclease form a complex and introduce a single- or double-stranded break in the sequence of the gene of interest. In some embodiments, the guide RNA is directed to a phytoene desaturase (PDS) gene. In some embodiments, the guide RNA is directed to a gene contributing to an agronomic trait of interest. In some embodiments, the gRNA is directed to a regulatory or coding sequence. In some embodiments, the regulatory or coding sequence contributes to a trait selected from the group consisting of: photosynthetic ability or efficiency; yield or fertility; seed number; disease or pest resistance; herbicide or pesticide tolerance; abiotic stressor tolerance; fruit morphology; fruit nutrition; fruit ripening; number of seeds per pod; and leaf size.

[0176] Chemical modifications to guide RNAs are known in the art, for example in U.S. Patent No. 10,337,001, and Ryan et al. 2018, Nucleic Acids Res. 46(20): 792-803.

[0177] In some embodiments 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. In some embodiments, 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. In some embodiments, 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.

[0178] In some embodiments, the nucleic acid encoding the guide RNA is linked to a promoter. In some embodiments, the promoter is an RNA polymerase II promoter or an RNA polymerase III promoter. In some embodiments, the RNA polymerase II promoter or RNA polymerase III promoter is endogenous to the species of the rootstock.

[0179] In some embodiments, a single guide RNA is provided to the plant. In other embodiments, multiple guide RNAs are provided to the plant. In some embodiments, the multiple guide RNAs are provided in a CRISPR array. In some embodiments, the two or more guide RNAs are encoded by a single precursor RNA. For the purposes of gene editing, CRISPR 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. In some embodiments, the CRISPR array comprises more than one spacer sequence. In some embodiments, the CRISPR array comprises more than one distinct spacer sequences. In some embodiments, the CRISPR array comprises more than one distinct spacer sequences designed to target the same genomic locus. In some embodiments, the CRISPR array comprises more than one distinct spacer sequences designed to target more than one distinct genomic loci. In some embodiments, the multiple guide RNAs are provided in a polycistronic system, wherein the multiple guide RNAs are 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 linked to multiple copies of the same promoter. In some embodiments, the multiple guide RNAs are 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 CRISPR array, wherein the CRISPR array is 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. In some embodiments, the multiple guide RNAs are provided in a polycistronic system, wherein the multiple guide RNAs are 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 linked to multiple copies of the same MTS. In some embodiments, the multiple guide RNAs are linked to different MTSs. [0180] In some embodiments, delivery of the guide RNA comprises spraying a composition comprising the guide RNA onto the leaves, shoot, stem, and/or meristem. In some embodiments, the composition comprising the guide RNA comprises a surfactant. In some embodiments, the composition comprising the guide RNA comprises glass beads coated with the guide RNA.

[0181] In some embodiments, delivery of the guide RNA comprises rubbing a composition comprising the guide RNA onto the leaves, shoot, stem, and/or meristem.

[0182] In some embodiments, delivery of the guide RNA comprises infecting the soybean plant with a viral vector.

[0183] In some embodiments, delivery of the guide RNA comprises infecting the soybean plant with a BPMV vector. In some embodiments, the BPMV comprises a MTS. In other embodiments, the BPMV does not comprise an MTS.

[0184] In some embodiments, delivery of the guide RNA comprises injecting a composition comprising the guide RNA into the stem.

[0185] In some embodiments, delivery of the guide RNA comprises leaf infiltration of a composition comprising the guide RNA into the leaf. In some embodiments, the leaf infiltration comprises forced infiltration using a needle-less syringe or vacuum pump.

[0186] In some embodiments, the guide RNA is delivered to the plant root by incubating the root with a composition comprising the guide RNA.

[0187] In some embodiments, 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.

[0188] In some embodiments, the guide RNA is delivered to the plant root by injecting a composition comprising the guide RNA into the root.

[0189] In some embodiments, the composition comprising the guide RNA comprises a nuclease inhibitor, optionally, wherein the nuclease inhibitor is an RNase inhibitor.

[0190] In some embodiments, the composition comprising the guide RNA comprises a nuclease inhibitor. In some embodiments, the nuclease inhibitor comprises an RNase inhibitor.

[0191] In some embodiments, application comprises biolistic transformation of nucleic acid encoding the guide RNA into the leaf, shoot, shoot, stem, and/or meristem. In some embodiments, the biolistic transformation comprises transformation of circular DNA encoding the guide RNA.

I. Donor Templates

[0192] In certain embodiments, a donor DNA template is provided in addition to the CRISPR Cas nuclease and the at least one gRNA, in order to effect incorporation of a DNA sequence from the donor DNA template at the target editing site in the plant genome by a mechanism such as, but not limited to, homology dependent repair (HDR), non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), or transgene integration. In some embodiments, the incorporation of a DNA sequence from the donor DNA template results in an insertion, deletion, and/or substitution in the DNA sequence at the target editing site in the plant genome.

[0193] In some embodiments, the virus-mediated delivery further comprises delivering a donor template DNA to the soybean plant, optionally wherein the donor template DNA is delivered by infecting the soybean plant with a viral vector that infects the meristem cell.

[0194] In some embodiments, the incorporation of a DNA sequence from the donor DNA template results in an insertion in the DNA sequence at the target editing site in the plant genome. In some embodiments, the incorporation of a DNA sequence from the donor DNA template results in a deletion in the DNA sequence at the target editing site in the plant genome. In some embodiments, the incorporation of a DNA sequence from the donor DNA template results in a substitution in the DNA sequence at the target editing site in the plant genome. Donor DNA template molecules used in the methods provided herein include DNA molecules comprising, from 5’ to 3’, a first homology arm, a replacement DNA, and a second homology arm, wherein the homology arms contain sequences that are partially or completely homologous to genomic DNA (gDNA) sequences flanking a target site in the genomic DNA. In some embodiments, the target editing site in the genomic DNA overlaps the site targeted by the gRNA. In certain embodiments, the replacement DNA can comprise an insertion, deletion, or substitution of one or more DNA base pairs relative to the target gDNA. In certain embodiments, the replacement DNA can comprise an insertion, deletion, or substitution of one or more DNA base pairs relative to the site targeted by the gRNA. In one embodiment, the donor DNA template molecule is double-stranded and perfectly base-paired through all or most of its length. In another embodiment, the donor DNA template molecule is double-stranded and includes one or more nonterminal mismatches or non-terminal unpaired nucleotides within the otherwise double-stranded duplex. In an embodiment, the donor DNA template molecule that is integrated at the site of at least one double-strand break (DSB) includes between 2-20 nucleotides in one (if single-stranded) or in both strands (if double-stranded), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides on one or on both strands, each of which can be base-paired to a nucleotide on the opposite strand of the targeted integration site (in the case of a perfectly base-paired double-stranded polynucleotide molecule). Such donor DNA templates can be integrated in genomic DNA containing blunt and/or staggered double stranded DNA breaks by a mechanism such as, but not limited to, homology dependent repair (HDR), non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), or transgene integration. In certain embodiments, a donor DNA template homology arm can be about 20, 50, 100, 200, 400, or 600 to about 800, or 1000 base pairs in length. In certain embodiments, a donor DNA template molecule can be delivered to a plant cell in a circular (e.g., a plasmid or a viral vector including a geminivirus vector or comovirus vector) or a linear DNA molecule. In certain embodiments, a circular or linear DNA molecule that is used can comprise a modified donor DNA template molecule comprising, from 5’ to 3’, a first copy of the target gRNA site sequence, the first homology arm, the replacement DNA, the second homology arm, and a second copy of the target gRNA site sequence. In other embodiments, DNA templates suitable for NHEJ insertion will lack homology arms that are partially or completely homologous to gDNA sequences flanking a target site-specific nuclease cleavage site in the gDNA. Compositions comprising the donor templates can be delivered to the plant and/or meristem cells of the plant by viral delivery, and other methods 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 cell-penetrating peptides. The donor template may be delivered by plasmid. The donor DNA templates may be present transiently in the cell or it could be introduced via a viral replicon (e.g., a gemini virus replicon). Gemini virus DNA replicons suitable for delivery of donor DNA templates to plants include a Beet Yellow Dwarf Virus replicon (Baltes, N.J. et al. Plant Cell vol. 26, 1 (2014): 151-63.). In some embodiments, the method further comprises delivering a donor template DNA to the plant by virus-mediated delivery. In some embodiments, a sequence from the donor template DNA is incorporated into the genome of the soybean plant. In some embodiments, the sequence is incorporated into the genome of the soybean plant at the genomic target. In some embodiments, a sequence from the donor template DNA is incorporated into the genome of the scion. In some embodiments, the donor template DNA is delivered to the scion using the same viral vector as the gRNA. In some embodiments, the donor template DNA is delivered to the soybean plant using the same viral vector as the gRNA. In some embodiments, the donor template DNA is delivered to the scion using a different viral vector than is used to deliver the gRNA. In some embodiments, the sequence from the donor template DNA is incorporated into the genome of the scion at the locus targeted by the gRNA. In some embodiments, the donor template DNA confers a desired trait. In some embodiments, the donor template comprises an endogenous sequence. In other embodiments, the donor template comprises an exogenous sequence. Donor templates can be utilized in VIGE alongside systems such as retron systems. In some embodiments, the method further comprises delivering a donor template DNA to the plant by virus-mediated delivery. In some embodiments, the donor template is delivered by infecting the soybean plant with a BPMV vector. In some embodiments, a sequence from the donor template DNA is incorporated into the genome of the soybean plant. In some embodiments, the sequence from the donor template DNA is incorporated into the genome of the soybean plant at the gene of interest. In some embodiments, the donor template is co-transfected with the guide RNA. In some embodiments, the donor template DNA is delivered to the soybean plant using a different viral vector than the viral vector carrying the guide RNA. In some embodiments, the donor template DNA is delivered to the soybean plant using a different BPMV vector than the BPMV vector carrying the guide RNA.

[0195] In some embodiments, the incorporation of a DNA sequence from the donor DNA template results in an insertion in the DNA sequence at the target editing site in the plant genome. In some embodiments, the incorporation of a DNA sequence from the donor DNA template results in a deletion in the DNA sequence at the target editing site in the plant genome. In some embodiments, the incorporation of a DNA sequence from the donor DNA template results in a substitution in the DNA sequence at the target editing site in the plant genome. Donor DNA template molecules used in the methods provided herein include DNA molecules comprising, from 5’ to 3’, a first homology arm, a replacement DNA, and a second homology arm, wherein the homology arms contain sequences that are partially or completely homologous to genomic DNA (gDNA) sequences flanking a target site in the genomic DNA. In some embodiments, the target editing site in the genomic DNA overlaps the site targeted by the gRNA. In certain embodiments, the replacement DNA can comprise an insertion, deletion, or substitution of one or more DNA base pairs relative to the target gDNA. In one embodiment, the donor DNA template molecule is double-stranded and perfectly base-paired through all or most of its length, with the possible exception of any unpaired nucleotides at either terminus or both termini. In another embodiment, the donor DNA template molecule is double-stranded and includes one or more non-terminal mismatches or non-terminal unpaired nucleotides within the otherwise double-stranded duplex. In an embodiment, the donor DNA template molecule that is integrated at the site of at least one double-strand break (DSB) includes between 2-20 nucleotides in one (if single-stranded) or in both strands (if double-stranded), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides on one or on both strands, each of which can be base-paired to a nucleotide on the opposite strand of the targeted integration site (in the case of a perfectly base-paired double-stranded polynucleotide molecule). Such donor DNA templates can be integrated in genomic DNA containing blunt and/or staggered double stranded DNA breaks by a mechanism such as, but not limited to, homology dependent repair (HDR), non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), or transgene integration. In certain embodiments, a donor DNA template homology arm can be about 20, 50, 100, 200, 400, or 600 to about 800, or 1000 base pairs in length. In certain embodiments, a donor DNA template molecule can be delivered to a plant cell in a circular (e.g., a plasmid or a viral vector including a geminivirus vector) or a linear DNA molecule. In certain embodiments, a circular or linear DNA molecule that is used can comprise a modified donor DNA template molecule comprising, from 5’ to 3’, a first copy of the target gRNA site sequence, the first homology arm, the replacement DNA, the second homology arm, and a second copy of the target gRNA site sequence. In other embodiments, DNA templates suitable for NHEJ insertion will lack homology arms that are partially or completely homologous to gDNA sequences flanking a target site-specific nuclease cleavage site in the gDNA. Compositions comprising the donor 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 cell-penetrating peptides. The donor DNA templates may be present transiently in the cell or it could be introduced via a viral replicon (e.g., a geminivirus replicon). Geminivirus DNA replicons suitable for delivery of donor DNA templates to plants include a Beet Yellow Dwarf Virus replicon (Baltes, N.J. et al. Plant Cell vol. 26, 1 (2014): 151-63.). In some embodiments, the method further comprises delivering a donor template DNA to the plant by virus- mediated delivery. In some embodiments, a sequence from the donor template DNA is incorporated into the genome of the scion. In some embodiments, the donor template DNA is delivered to the scion using the same viral vector as the gRNA. In some embodiments, the donor template DNA is delivered to the scion using a different viral vector than is used to deliver the gRNA. In some embodiments, the sequence from the donor template DNA is incorporated into the genome of the scion at the locus targeted by the gRNA. In some embodiments, the donor template DNA confers a desired trait. In some embodiments, the donor template comprises an endogenous sequence. In other embodiments, the donor template comprises an exogenous sequence.

J. Prime Editing

[0196] Desired DNA sequence modifications can be accomplished through the use of PRIME editing (Anzalone et al. Nature 2019, 576(7785): 149-157). In some embodiments, prime editing uses (i) a Cas nickase, in some embodiments a Cas9 nickase, in other embodiments a Casl2 nickase, fused to a reverse transcriptase (nCas-RT), in some embodiments a M-MLV reverse transcriptase, and (ii) a prime editing Cas guide RNA (pegRNA) that both specifies the genome target site and has an extension that encodes the target edit within a template for the reverse transcriptase. In some embodiments, gRNA is a prime editing guide RNA (pegRNA). The binding of the pegRNA directs the Cas nickase to create a singlestranded break in the DNA at the nicking site. The extension of the pegRNA binds to the nicked DNA that has an exposed 3 ’-hydroxyl group, priming the reverse transcriptase to produce a DNA strand that is complementary to the extension of the pegRNA. This DNA strand will include the complement to any desired edits present in the provided pegRNA extension. Mismatch repair by the cell will then resolve the mismatch between the unedited parent strand and the edited product of the reverse transcriptase, thus introducing the desired edits into the genome. Prime editing systems may also include elements to inhibit mismatch repair, or to nick the unedited parent strand to increase editing efficiency. A mobility element can be fused to the pegRNA so as not to interfere with priming of the reverse transcriptase. In some embodiments of a viral vector system for use in soybean editing, the system comprises a plant virus genome component; one or more pegRNA; and a Cas nuclease fused to a reverse transcriptase. In some embodiments, the donor template DNA is delivered to the soybean plant using a different viral vector than the viral vector comprising the gRNA. In some embodiments, the donor template DNA confers a desired trait on the plant. In some embodiments, the donor template is cotransfected with the guide RNA. In some embodiments, the donor template DNA is delivered to the soybean plant using a different viral vector than the viral vector carrying the guide RNA. In some embodiments, the donor template comprises an exogenous sequence. In some embodiments, the donor template comprises an endogenous sequence. [0197] Desired DNA sequence modifications can be accomplished through the use of PRIME editing (Anzalone et al. Nature 2019, 576(7785): 149-157). In some embodiments, prime editing uses (i) a Cas nickase, in some embodiments a Cas9 nickase, in other embodiments a Casl2 nickase, fused to a reverse transcriptase (nCas-RT), in some embodiments a M-MLV reverse transcriptase, and (ii) a prime editing Cas guide RNA (pegRNA) that both specifies the genome target site and has an extension that encodes the target edit within a template for the reverse transcriptase . The binding of the pegRNA directs the Cas nickase to create a single-stranded break in the DNA at the nicking site. The extension of the pegRNA binds to the nicked DNA that has an exposed 3 ’-hydroxyl group, priming the reverse transcriptase to produce a DNA strand that is complementary to the extension of the pegRNA. This DNA strand will include the complement to any desired edits present in the provided pegRNA extension. Mismatch repair by the cell will then resolve the mismatch between the unedited parent strand and the edited product of the reverse transcriptase, thus introducing the desired edits into the genome. Prime editing systems may also include elements to inhibit mismatch repair, or to nick the unedited parent strand to increase editing efficiency. A mobility element can be fused to the pegRNA so as not to interfere with priming of the reverse transcriptase.

[0198] In some embodiments, prime editing can also be accomplished with Cas nucleases in place of Cas nickases (Adikusuma et al. Nucleic Acids Res. 2021, 49(18): 10785-10795). In some embodiments, prime editing uses (i) a Cas nuclease, in some embodiments a Cas9 nuclease, in other embodiments a Casl2 nuclease, fused to a reverse transcriptase (Cas-RT), in some embodiments a M-MLV reverse transcriptase, and (ii) a prime editing Cas guide RNA (pegRNA) that both specifies the genome target site and has an extension that encodes the target edit within a template for the reverse transcriptase. In some embodiments, the binding of the pegRNA directs the Cas nuclease to create a double-stranded break in the DNA at the target site. The extension of the pegRNA binds to the cut DNA that has an exposed 3 ’-hydroxyl group, priming the reverse transcriptase to produce a DNA strand that is complementary to the extension of the pegRNA. This DNA strand will include the complement to any desired edits present in the provided pegRNA extension. Mismatch repair by the cell will then resolve the mismatch between the unedited parent strand and the edited product of the reverse transcriptase, thus introducing the desired edits into the genome. Prime editing systems may also include elements to inhibit mismatch repair, or to nick the unedited parent strand to increase editing efficiency. A mobility element can be fused to the pegRNA so as not to interfere with priming of the reverse transcriptase.

[0199] Prime editing makes precise DNA sequence modifications rather than random insertions, deletions, and substitutions (Indels), thus increasing the probability of obtaining the desired effect. Prime editing may be used to introduce any single base pair substitution as well as small deletion or insertions. Deletions of up to 80 base pairs have been produced using prime editing with a single pegRNA in human cells, and insertions of up to 40 base pairs (Anzalone et al. Nature 2019, 576: 149- 157). Dual pegRNA systems are also known in the art (Choi et al. Nat Biotechnol 2021, 40(2): 218- 226; Lin et al. Nature Biotechnology 2021, 39(8): 923-927) and can be used to generate precise large deletions, or to improve editing efficiency for small insertions, deletions, or substitutions. Additionally, dual pegRNA systems where the extension of the pegRNAs are not complementary to the endogenous locus, but are complementary to one another, can be used to replace endogenous sequence and/or mediate larger insertions (Anzalone et al. Nat Biotechnol 2022, 40(5): 731-740).

[0200] In some embodiments, the Cas nuclease is associated with a reverse transcriptase. In some embodiments, the Cas nuclease is fused to the reverse transcriptase. In some embodiments, the guide RNA comprises at its 3’ end a priming site and an edit to be incorporated into the genomic target. In some embodiments, the Cas nuclease is a Cas nickase. 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.

J. Bean Pod Mottle Virus (BPMV)

[0201] In some embodiments, the methods provided herein involve delivery of one or more components of a gene editing systems (e.g., a guide RNA and/or a Cas enzyme) to modify or edit a soybean plant meristem cell. In some aspects of the present disclosure, a viral vector is used to transport one or more components of a gene editing system (e.g., a guide RNA and/or a Cas enzyme). In some aspects of the present disclosure, bean pod mottle virus (BPMV) is used to transport one or more components of a gene editing system (e.g., a guide RNA and/or a Cas enzyme). In some embodiments, the viral vector comprises bean pod mottle virus (BPMV).

[0202] In some embodiments, the viral vector comprises a recombinant plant virus that comprises the guide RNA or a polynucleotide encoding the guide RNA. In some embodiments, the recombinant plant virus used in the virus-mediated delivery is a virus with a segmented genome. The BPMV genome requires both bipartite genome segments RNA1 and RNA2 for infection, but these segments may be delivered separately or in union. In some embodiments, the BPMV vector comprises RNA2. Sequence information regarding genome segments RNA 1 and RNA 2 is publicly available (such as in, for example, Zhang, C. et al. The development of an efficient multipurpose Bean pod mottle virus viral vector set for foreign gene expression and RNA silencing. Plant Physiol. 2010, 153(l):52-65; Pflieger, S, et al. The "one-step" Bean pod mottle virus (BPMV)-derived vector is a functional genomics tool for efficient overexpression of heterologous protein, virus-induced gene silencing and genetic mapping of BPMV R-gene in common bean (Phaseolus vulgaris L.). BMC Plant Biol. 2014, Aug 29; 14:232). In some embodiments, the BPMV-RNA2 is linked to, or otherwise carries, the gRNA. The gRNA sequence may be inserted into nonessential portions of the BPMV-RNA2 genomic sequence. The gRNA sequence may be linked to the BPMV-RNA2 sequence by processing elements. In some embodiments, the BPMV-RNA2 is linked to, or otherwise carries, the gRNA. In some embodiments, the BPMV-RNA1 is linked to, or otherwise carries, the gRNA. The gRNA sequence may be inserted into nonessential portions of the BPMV-RNA1 genomic sequence. The gRNA sequence may be linked to the BPMV-RNA1 sequence by processing elements. The disclosure herein comprises kits comprising the viral vector systems described herein, as well as a manual for utilizing said kits. K. Delivery to the Meristem

[0203] In some embodiments, the methods provided herein involve transport of one or more components of a gene editing systems (e.g., a Cas nuclease and a guide RNA) to the meristem. 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. For example, Arabidopsis FT-based vectors work in Nicotiana benthamiana and Arabidopsis. Vectors can also be designed based on alternative sequences, which can be based either on the species subject to genomic editing or based on a different species, sometimes a related species, sometimes a closely related species.

[0204] While 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.

[0205] The Flowering Locus T (FT) mRNA is useful as a meristem transport segment. SEQ ID NO: 2 shows the DNA sequence that encodes the Arabidopsis FT RNA, and 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 (Sun et al. PLoS One. 2011, 6(12): e29238. doi:10[dot]1371/journal[dot]pone[dot]0029238; Jiang et al. BMC Genomics. 2019 20(1): 230. doi: 10[dot] 1186/sl2864-019-5577-5; Kong et al. Plant Physiol. 2010 Nov, 154(3): 1220-31. doi: 10[dot]1104/pp[dot]110[dot] 160796; Takeshima et al. J Exp Bot. 2019 Aug 7, 70(15): 3941-3953. doi: 10[dot]1093/jxb/erzl99), which may work across related dicot species. 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. Plant Cell 2016, 28: 1237-1249), variants thereof, and fragments thereof. 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.

[0206] More generally, viral and cellular-derived RNA molecules that are useful as part of a transport segment include the mRNAs of FT, GAI, CmNACP, tomato LeT6, a KNOX gene, BEL5, 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 Li 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). GAI 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. 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. 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. Examples of tRNA-like RNAs that can be used 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. In certain embodiments, 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[dot]org/10[dot]1105/tpc[dot]15[dot]01056.

[0207] 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. Further description of biological sequences provided in the sequence listing is set forth in Table 2.

Table 1. Description of biological sequences.

Table 2. Description of further biological sequences

[0208] The choice of viral vector for use in VIGE is highly dependent on the species to be edited, due to the high specificity associated with the pathogen-host relationships. The viral vector itself determines the success of VIGE’s invasion of targeted meristems and meristem cells, rather than associated mobility elements (Beernink B. et al. Impacts of RNA Mobility Signals on Virus Induced Somatic and Germline Gene Editing. Frontiers in Genome Editing. 2022 4: 2673-3439). Selecting a virus as a viral vector also requires a balance between the virus’s capacity to infect a plant and the plant’s capacity to tolerate the virus. Alongside compatibility with a plant to be modified, another critical aspect of choosing a suitable viral vector is the size of the cargo that the vector needs to carry. Viral vectors are often very limited in the available space for inserting genome editing reagents (i.e., a guide RNA and/or a Cas enzyme), and the limited cargo space often challenges the virus vector’s ability to incorporate Cas enzyme components or, potentially, a donor template. One advantage of the present disclosure is that BPMV can carry relatively large inserts, with estimated RNA2 vector capacity between 1.4 - 1.8 kb in length (Zhang et al. The development of an efficient multipurpose Bean pod mottle virus viral vector set for foreign gene expression and RNA silencing. Plant Physiol. 2010. 153: 52-65). For more detail on selecting and engineering viral vectors, see, for example, Wu et al. 2024 (Wu, X. et al. Considerations in engineering viral vectors for genome editing in plants, Virology. 2024. Volume 589. 109922, ISSN 0042-6822).

[0209] 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. The RNA sequences can be expressed in vitro and introduced into the phloem as substantially purified molecules. Producing inoculum and/or thoughtful selection and preparation of recipient plant tissue can greatly increase the levels of success for infecting a soybean plant with a viral vector or composition comprising a recombinant plant virus. For example, 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, meristem-associated tissue, or any vegetative tissue. RNAs can be coated on particles, such as micro or nano-scale particles such as gold or tungsten, for biolistic delivery. In some embodiments, these RNA molecules include one or both genomic segments of the BPMV genome, RNA1 and RNA2. Alternatively, the guide RNA sequences can 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. 2018, 244: 333-337; Gao et al. New Phytol. 2019, 223: 2120-2133) or the MTC 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. Ideal viral vector 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. A clear readout of successful delivery to the meristem and modification of the meristem 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. A clear readout of successful delivery to the meristem cell and modification of the meristem cell 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. For example, 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 as a result of photobleaching of the mutant serving as a readout (see, for example, Kumagai et al. PNAS 1995, 92(5): 1679-1683; Xie et al. PNAS 2015, 112(11): 3570-3575).

[0210] In some embodiments, the meristem transport segment (MTS) 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. In some embodiments, 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. In some embodiments, the TLS comprises the nucleotide sequence set forth in SEQ ID NO: 29 or 30.

[0211] In some embodiments, the nucleic acid encoding the MTS is located 3’ of the nucleic acid encoding the Cas 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 Cas nuclease and/or 5’ of the nucleic acid encoding the guide RNA. In some embodiments, the nucleic acid encoding the MTS is located 3’ of the nucleic acid encoding the Cas nuclease and/or 3’ of the nucleic acid encoding the guide RNA.

[0212] In some embodiments, the nucleic acid encoding the BPMV genomic segment is located 5’ of nucleic acid encoding the Cas nuclease and/or 5’ of the nucleic acid encoding the guide RNA. In some embodiments, the nucleic acid encoding the guide RNA is located 5’ of the nucleic acid encoding the Cas nuclease. In some embodiments, the nucleic acid encoding the guide RNA is located 5’ of the nucleic acid encoding the guide RNA. In some embodiments, the nucleic acid encoding the BPMV genomic segment is located 3’ of nucleic acid encoding the Cas nuclease and/or 5’ of the nucleic acid encoding the guide RNA. In some embodiments, the nucleic acid encoding the guide RNA is located 3’ of the nucleic acid encoding the Cas nuclease. In some embodiments, the nucleic acid encoding the guide RNA is located 3’ of the nucleic acid encoding a second guide RNA. In some embodiments, nucleic acid encoding the BPMV genomic segment is located 5’ and/or 3’ of nucleic acid encoding the Cas nuclease. In some embodiments, nucleic acid encoding the BPMV genomic segment is located 5’ and/or 3’ of nucleic acid encoding the guide RNA.

[0213] In some embodiments, the plant further comprises nucleic acid encoding a detectable marker fused to a nucleic acid encoding an MTS.

L. Genome Modifications

[0214] The reagents and methods described provide a relatively easy and convenient solution for producing plants with altered genomes, i.e., individuals with designed DNA sequence modifications (e.g., Indels or epigenetic alterations). 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). In some embodiments, the edit results in the insertion or deletion of nucleotides at or near the target sequence. In some embodiments, the edit results in an insertion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40 nucleotides at or near the target sequence. In some embodiments, the edit results in a deletion of at least 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 at or near the target sequence. In some embodiments, the edit results in a nucleotide substitution at or near the target sequence. In some embodiments, the edit results in a substitution of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides at or near the target sequence. In most embodiments, 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., Indels or epigenetic alterations) are carried into germline cells and subsequent generations.

[0215] Very often, mutated seeds from plants edited with the reagents and methods described here are collected for phenotypic characterization. In some cases, pollen from edited plants is used in crosses with other individuals, or mutated individuals are pollinated with pollen of unedited plants or wildtype plants. In some embodiments, the method of editing a genomic target in a meristem cell of a soybean plant comprises virus-mediated delivery, wherein the virus-mediated delivery comprises infecting the soybean plant with a bean pod mottle virus (BPMV) vector carrying a guide RNA (gRNA) directed to the genomic target in the meristem cell of the soybean plant. In some embodiments, the method further comprises screening the soybean plant for viral infection, said screening comprising a visual assessment of the soybean plant for a desired phenotype. In some embodiments, the method further comprises screening the soybean plant for successful genome modification, said screening comprising visually assessing the soybean plant for desired phenotype. In some embodiments, said screening further comprises sequencing of cells produced by the meristem after delivery of the BPMV vector. In some embodiments, the method further comprises screening the progeny of the soybean plant for successful genome modification, said screening comprising visually assessing a soybean plant that grows from the seed for desired phenotype, and/or sequencing of cells.

[0216] 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) is also eliminated. The editing can be performed in individuals of an elite genetic background, making lengthy backcrossing schemes unnecessary.

[0217] Plants comprising the RNA molecules that comprise a Cas nuclease and/or guide RNA(s) are also provided herein. Plants comprising the RNA molecules that comprise a Cas nuclease and/or guide RNA(s) that are linked to MTS sequences are also provided herein. In certain embodiments, such RNA molecules will be present at detectable concentrations in the plants for only a certain period of time following a stimulus. For example, 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)) which are capable of being processed (i.e., cleaved) by an RNA- guided nuclease are expected to decrease over time when the RNA-guided nuclease is also present in the plant. The 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. Nonetheless, the unprocessed 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 linked to MTS sequences are used. The unprocessed RNA molecules can also 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 viral vector, Cas nuclease, and/or guide RNA(s) are used. 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. In certain embodiments, an active form of the RNA guided nuclease is predominantly localized in meristem tissue of the plant. In certain embodiments, an active form of the RNA guided nuclease is predominantly localized in meristem cells of the plant. In certain embodiments, the RNA- guided nuclease can be encoded by an RNA molecule that optionally further comprises a linked MTS sequence. In certain embodiments, the RNA-guided nuclease can be encoded by an RNA molecule that optionally further comprises a viral vector. In certain embodiments, the RNA-guided nuclease can be encoded by DNA that is linked to promoters that include a root-preferred or root-specific promoter which is active in root cells. In certain embodiments, the RNA-guided nuclease can be encoded by DNA that is 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 [dot] 1105/tpc [dot] 113 [dot] 119792).

[0218] Nonetheless, the unprocessed 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 viral vector, Cas nuclease and/or guide RNA(s) are used. 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. In certain embodiments, an active form of the RNA guided nuclease is predominantly localized in meristem cells of the plant. In certain embodiments, the RNA-guided nuclease can be encoded by an RNA molecule that optionally further comprises an linked MTS sequence. In certain embodiments, the RNA-guided nuclease can be encoded by DNA that is linked to promoters that include a root-preferred or root-specific promoter which is active in root cells. In certain embodiments, the RNA-guided nuclease can be encoded by DNA that is 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[dot]1105/tpc[dot] 113 [dot] 119792). In certain embodiments, the RNA-guided nuclease can be encoded by an RNA molecule that optionally further comprises a viral vector.

[0219] Plants comprising the RNA molecules that comprise a Cas nuclease and/or guide RNA(s) that are linked to MTS sequences are also provided herein. Also provided herein are plants that comprise the RNA molecules that comprise a Cas nuclease and/or guide RNA(s) that are linked to MTS sequences. In certain embodiments, such RNA molecules will be present at detectable concentrations in the plants for only a certain period of time following a stimulus. In some embodiments, viral vectors delivered as DNA are then processed into RNA by transcription. In certain embodiments, an MTS is linked to a CRISPR Cas system 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. In certain embodiments, a viral vector comprises a linked CRISPR Cas system 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. In certain embodiments, 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. Examples of such 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). Cleavage of RNAs comprising Casl2a DRs by Casl2a has been described (Fonfara et al. Nature 2016, 532: 517-521, doi[dot]org/10[dot] 1038/naturel7945); US20160208243; WO 2017/189308). Other examples of such DRs include the Casl2j DRs (e.g., SEQ ID NO: 58, 60, or 62) which can be cleaved by a Casl2j guided nuclease (e.g., SEQ ID NO: 57, 59, or 61, respectively). In such embodiments, 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.

[0220] In some embodiments, the meristem is part of a plant scion grafted onto a rootstock. In other embodiments, the meristem is part of a non-grafted plant.

II. Targets of Genomic Modification

[0221] 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. Embodiments of the viral vector system, BPMV viral vector system, BPMV vector, 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. In embodiments, the target sequence or target gene includes coding sequence (DNA encoding a polypeptide, such as a structural protein or an enzyme), non-coding sequence, or both coding and non-coding sequence. Embodiments of the polynucleotides, compositions, engineered systems, BPMV vector systems, other viral vector 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. In embodiments, the target sequence or target gene includes coding sequence (DNA encoding a polypeptide, such as a structural protein or an enzyme), non-coding sequence, or both coding and non-coding sequence.

A. Identification of Targets

[0222] There are numerous plant-endogenous targets (i.e., DNA sequence targets) for genome editing. The methods presented here 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 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).

[0223] The method of the present invention may be used to introduce edits to affect any phenotype, quality, or trait of the organism. For instance, 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. In some embodiments, the viral vector carries at least one guide RNA. In some embodiments, the BPMV vector carries at least one guide RNA. In some embodiments, the guide RNA is directed to a regulatory or coding sequence contributing to trait selected from the group consisting of: photosynthetic ability or efficiency; yield or fertility; seed number; disease or pest resistance; herbicide or pesticide tolerance; abiotic stressor tolerance; fruit morphology; fruit nutrition; fruit ripening; number of seeds per pod; and leaf size. In some embodiments, the guide RNA comprises a spacer that is complementary to a target sequence. In some embodiments, the target sequence is located within a target gene or a target genomic region.

[0224] 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). For example, a target gene may be selected that has a current, baseline level of expression in a target plant species. Guide RNAs may be produced that target different regions of the promoter of this target gene. Multiple lines of the elite germplasm may be generated containing distinct edits in the target gene promoter using the methods provided herein. For example, one line may have deleted a transcription factor binding site; a second line may have introduced 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. In some embodiments, 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.

[0225] Any defective, deleterious, non-optimal, or underperforming allele found in elite germplasm can be edited to a non-deleterious or more optimal allele. In some embodiments, a target to be modified is a genetic variant that is known in the art to be deleterious. In some embodiments, 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). In some embodiments, 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. Chun and Fay Genome Res 2009, 19: 1553-1561; Rodgers-Melnick et al. PNAS 2015, 112: 3823-3828), functional impact of amino acid change (See e.g. Ng et al. NAR 2003, 31: 3812-3814; Adzhubei et al. Nat Methods 2010, 7: 248-249), functional impact of protein conformation and/or stability (See e.g. Rosetta, a computational protein design platform from Cyrus Bio Inc.), adjacency to selective sweep regions (See e.g. Hufford et al. Nat Gen 2012, 44: SOS- 813), and outlier status of a 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 (See e.g. Zhao et al. AJHG 2016, 98: 299-309).

[0226] Editing of coding sequences can be made using the methods disclosed herein to increase the level of preselected amino acids in the encoded polypeptide. For example, 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); and rice (Musumura et al. Plant Mol. Biol. 1989, 12: 123, herein incorporated by reference). Other agronomically important genes encode latex, Floury 2, growth factors, seed storage factors, and transcription factors.

[0227] 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. 20040082770 and WO 03/092360); or other such genes known in the art. The bar gene encodes resistance to the herbicide basta, the nptll gene encodes resistance to the antibiotics kanamycin and geneticin, and the ALS-gene mutants encode resistance to the herbicide chlorsulfuron. Additional herbicide resistance traits are described for example in U.S. Patent Application 2016/0208243, herein incorporated by reference.

[0228] 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.

[0229] Genome editing can also be used to make haploid inducer lines as disclosed in WO2018086623 and US20190292553.

[0230] 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. In corn, modified hordothionin proteins are described in U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389.

[0231] Commercial traits can also be altered by modifying a gene or that could increase for example, starch for ethanol production, or provide expression of proteins. Another important commercial use of modified plants is the production of polymers and bioplastics such as described in U.S. Pat. No. 5,602,321. Genes such as beta- Ketothiolase, PHBase (polyhydroxyburyrate synthase), and acetoacetyl- CoA reductase (see Schubert et al. J. Bacteriol 1988, 170: 5837-5847) facilitate expression of polyhyroxy alkanoates (PH As).

[0232] 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

Ill 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.

[0233] 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. Such 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. Genes capable of conferring these desirable traits are disclosed in U.S. Patent Application 2016/0208243, herein incorporated by reference.

[0234] In some embodiments, 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. In some embodiments, 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.

B. Plants

[0235] The present disclosure may be used for genomic editing of any soybean plant species, including, but not limited to, cultivated soybean Glycine max) and wild soybean (G. soja, or G. max subsp. Soja (Siebold & Zucc.) H. Ohashi). Examples of soybean plant species of interest include, but are not limited to, species and subspecies of Glycine subgenus Soja (Moench) F.J. Herm. and Glycine subgenus Glycine. Further examples include, but are not limited to, soybean species Glycine arenaria, Glycine argyrea, Glycine canescens, Glycine clandestina, Glycine curvata, Glycine cyrtoloba, Glycine falcata, Glycine latifolia, Glycine latrobeana, Glycine max (E.) Merr, Glycine microphylla, Glycine pindanica, Glycine priceana, Glycine tabacina, Glycine tomentella, and Glycine wrightii.

[0236] In some embodiments, the plant is edited with VIGE. In some embodiments, the method edits a genomic target in a soybean plant, the method comprising: delivering a guide RNA (gRNA) directed to the genomic target in the meristem cell in the soybean plant by virus-mediated delivery; and delivering a Cas nuclease to the meristem cell, wherein the Cas nuclease and the guide RNA edit the genomic target in the meristem of the soybean plant, thereby editing the genomic target in the meristem cell. Another aspect of the present disclosure provides a soybean plant produced by the methods herein, wherein the produced soybean plant comprises the edited genomic target.

[0237] Another aspect of the present disclosure provides a method of producing a soybean seed comprising an edited genomic target, the method comprising: delivering a guide RNA (gRNA) directed to the genomic target in a meristem cell of a parent soybean plant by virus-mediated delivery; and delivering a Cas nuclease to the meristem cell of the parent soybean plant; wherein the Cas nuclease and the guide RNA edit the genomic target in the meristem cell of the parent soybean plant, and wherein the meristem cell produces a soybean germline cell that contributes to the soybean seed, and thereby producing the soybean seed comprising the edited genomic target. Also provided in the present disclosure is a method for producing a soybean meristem cell having an edited genomic target, the method comprising: delivering a viral vector carrying a gRNA to a soybean meristem cell, wherein the soybean meristem cell comprises a Cas nuclease; allowing the gRNA and the Cas nuclease to modify the soybean meristem cell; and thereby producing the soybean meristem cell having the edited genomic target. In some embodiments, the edited genomic target is inherited by at least one progeny or seed of the soybean plant. In some embodiments, the method further comprises allowing the meristem cell to generate a seed comprising the edited genomic target and collecting the seed. In some embodiments, the method further comprises growing the seed. In some embodiments, the method further comprises retrieving a progeny of the scion, wherein the progeny comprises the edited genomic target. Another aspect of the present disclosure is a soybean seed produced by the methods herein, wherein the produced soybean seed comprises the edited genomic target. Also provided is a soybean meristem cell produced by the methods herein, wherein the soybean meristem cell comprises the edited genomic target. In some embodiments of the present disclosure, the progeny does not inherit the guide RNA and/or the Cas enzyme.

[0238] In some embodiments of the present disclosure, a method for producing a meristem cell having a targeted genomic modification is provided. The method comprises delivering a BPMV viral vector carrying a guide RNA to a meristem cell, wherein the meristem cell expresses a Cas enzyme; allowing the gRNA and the Cas enzyme to modify the meristem cell; and thereby producing the meristem cell having the targeted genomic modification. Also provided is a meristem cell having the modification produced by the methods herein. In some embodiments, a method for producing soybean seed comprising a targeted genomic modification is provided. The method comprises delivering a BPMV viral vector carrying a gRNA to a soybean meristem cell, wherein the soybean meristem cell expresses a Cas nuclease; wherein the gRNA and the genomic modification enzyme modifies the soybean meristem cell; wherein the soybean meristem cell produces a soybean germline that forms seed; and thereby producing soybean seed having the targeted genomic modification. Also provided is a seed comprising an inherited modification in a gene of interest in a soybean plant, the seed produced by the methods herein. In some embodiments, the seed does not comprise the guide RNA and/or the Cas enzyme.

[0239] 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). Examples of plant species of interest include, but are not limited to, corn (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 cere ale), sorghum (Sorghum bicolor, Sorghum vulgare), camelina (Camelina sativa), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum 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 batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.). avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oil palm (Elaeis guineensis), poplar (Populus spp.), eucalyptus (Eucalyptus spp.), oats (Avena sativa), barley (Hordeum vulgare), sesame (Sesamum spp.), flax (Linum usitatissimum), cannabis (Cannabis spp.), a vegetable crop, a forage crop, an industrial crop, a woody crop, a biomass crop, an ornamental, and a conifer.

[0240] In some embodiments, 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, corn, oat, sorghum, sugarcane, banana, or wheat.

[0241] In some embodiments, the meristem is edited. In some embodiments, the genome of a meristem of a plant scion grafted onto a rootstock is edited. In some embodiments, the meristem cell is edited. In some embodiments, the genome of a meristem cell of a plant scion grafted onto a rootstock is edited.

III. Delivery

A. Vectors

[0242] Vectors are used to deliver nucleic acids to plant cells. In some embodiments, the vector is capable of autonomous replication within the host cell. In other embodiments, the vector is integrated into the genome of the host cell and replicated with the host genome. In some embodiments, termed “expression vectors”, the genes of the vector are expressed or are capable of being expressed under certain conditions. In some embodiments, the vector contains one or more regulatory elements linked to a gene. In some embodiments, the vector contains a promoter. In some embodiments, 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). In some embodiments, 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.

[0243] In some embodiments of the method, the nucleic acid described herein can contained within any suitable plant transformation plasmid or vector. In some embodiments, the plant transformation plasmid or vector further comprises a selectable or screenable marker, such as but not limited to a fluorescent protein or an herbicide -resistance protein. In some embodiments, the recombinant plant virus provided herein further comprises an expression cassette comprising an endogenous visible marker gene or a reporter gene, optionally wherein the reporter gene encodes a fluorescent reporter.

[0244] In some embodiments of the present disclosure, provided is a method of editing a genomic target in a meristem cell a soybean plant, the method comprising delivering a guide RNA (gRNA) directed to the genomic target to the meristem cell in the soybean plant by a viral vector; and delivering a Cas nuclease to the meristem cell, wherein the Cas nuclease and the guide RNA edit the genomic target in the meristem cell of the soybean plant, thereby editing the genomic target in the meristem cell;. In some embodiments of the method, the gRNA and/or the Cas nuclease is contained within a bean pod mottle virus (BPMV) vector.

[0245] Also provided in some aspects of the present disclosure is a bean pod mottle virus (BPMV) viral vector system comprising: a BPMV genome component, one or more gRNA inserted into the viral vector, and a direct repeat and/or self-cleaving ribozyme sequence flanking the one or more gRNA. In some embodiments, a nucleic acid modifying enzyme is overexpressed in cells that receive the viral vector system. In some embodiments, the viral vector system further comprises a nucleic acid encoding a nucleic acid modifying enzyme. In some embodiments, the nucleic acid modifying enzyme is a CRISPR/Cas nuclease.

[0246] In embodiments of the method, the component(s) of a gene editing system are delivered via at least one viral vector, including comoviruses. Suitable comovirus vectors include, for example, a bean pod mottle virus (BPMV) vector and the like. In some embodiments, the recombinant plant virus used in the virus-mediated delivery is a positive strand RNA virus. In embodiments of the method, 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, gemini viruses, begomoviruses, tobamoviruses, potexviruses, potyviruses, tobraviruses, tombusviruses, bromoviruses, carmoviruses, alfamoviruses, cucumo viruses, comoviruses, and hordeviruses. See, e.g., Peyret and Lomonossoff Plant Biotechnol. J. 2015, 13:1121. Suitable tobamovirus vectors include, for example, a tomato mosaic virus (ToMV) vector, a tobacco mosaic virus (TMV) vector, a tobacco mild green mosaic virus (TMGMV) vector, a pepper mild mottle virus (PMMoV) vector, a paprika mild mottle virus (PaMMV) vector, a cucumber green mottle mosaic virus (CGMMV) vector, a kyuri green mottle mosaic virus (KGMMV) vector, a hibiscus latent fort pierce virus (HLFPV) vector, an odontoglossum ringspot virus (ORSV) vector, a rehmannia mosaic virus (ReMV) vector, a Sammon's opuntia virus (SOV) vector, a wasabi mottle virus (WMoV) vector, a youcai mosaic virus (YoMV) vector, a sunn-hemp mosaic virus (SHMV) vector, and the like. Suitable Potexvirus vectors include, for example, a potato virus X (PVX) vector, a potato aucuba mosaic virus (PAMV) vector, an Alstroemeria virus X (AlsVX) vector, a cactus virus X (CVX) vector, a Cymbidium mosaic virus (CymMV) vector, a hosta virus X (HVX) vector, a lily virus X (LVX) vector, a Narcissus mosaic virus (NMV) vector, a Nerine virus X (NVX) vector, a Plantago asiatica mosaic virus (PIAMV) vector, a strawberry mild yellow edge virus (SMYEV) vector, a tulip virus X (TVX) vector, a white clover mosaic virus (WC1MV) vector, a bamboo mosaic virus (BaMV) vector, a foxtail mosaic virus (FoMV) vector, and the like. Suitable Potyvirus vectors include, for example, a wheat streak mosaic virus (WSMV), a potato virus Y (PVY) vector, a bean common mosaic virus (BCMV) vector, a clover yellow vein virus (C1YVV) vector, an East Asian Passiflora virus (EAPV) vector, a Freesia mosaic virus (FreMV) vector, a Japanese yam mosaic virus (JYMV) vector, a lettuce mosaic virus (LMV) vector, a Maize dwarf mosaic virus (MDMV) vector, an onion yellow dwarf virus (OYDV) vector, a papaya ringspot virus (PRSV) vector, a pepper mottle virus (PepMoV) vector, a Perilla mottle virus (PerMo V) vector, a plum pox virus (PPV) vector, a potato virus A (PVA) vector, a sorghum mosaic virus (SrMV) vector, a soybean mosaic virus (SMV) vector, a sugarcane mosaic virus (SCMV) vector, a tulip mosaic virus (TulMV) vector, a turnip mosaic virus (TuMV) vector, a watermelon mosaic virus (WMV) vector, a zucchini yellow mosaic virus (ZYMV) vector, a tobacco etch virus (TEV) vector, and the like. Suitable Tobravirus vectors include, for example, a tobacco rattle virus (TRV) vector and the like. Suitable Tombusvirus vectors include, for example, a tomato bushy stunt virus (TBSV) vector, an eggplant mottled crinkle virus (EMCV) vector, a grapevine Algerian latent virus (GALV) vector, and the like. Suitable Cucumovirus vectors include, for example, a cucumber mosaic virus (CMV) vector, a peanut stunt virus (PSV) vector, a tomato aspermy virus (TAV) vector, and the like. Suitable Bromovirus vectors include, for example, a brome mosaic virus (BMV) vector, a cowpea chlorotic mottle virus (CCMV) vector, and the like. Suitable Carmovirus vectors include, for example, a carnation mottle virus (CarMV) vector, a melon necrotic spot virus (MNSV) vector, a pea stem necrotic virus (PS NV) vector, a turnip crinkle virus (TCV) vector, and the like. Suitable Alfamovirus vectors include, for example, an alfalfa mosaic virus (AMV) vector, and the like. Suitable Comovirus vectors include, for example, a bean pod mottle virus (BPMV) vector, a cowpea mosaic virus (CPMV) vector, and the like. Suitable Hordevirus vectors include, for example, a barley stripe mosaic virus (BSMV) vector, and the like. Suitable Begomovirus vectors include, for example, a cabbage leaf curl virus (CabLCV) vector, a soybean mild mottle virus (SbMMV) vector, and the like. Suitable Geminivirus vectors include, for example, a bean yellow dwarf virus (BeYDV) vector, a beet curly top virus (BCTV) vector, a tobacco yellow dwarf virus (TYDV) vector, and the like. In some embodiments, the recombinant plant virus used in the virus-mediated delivery is a positive strand RNA virus. In some embodiments, the Cas nuclease is delivered by virus-mediated delivery. In some embodiments, the viral vector comprises the Cas nuclease. In some embodiments, the viral vector comprising the gRNA further comprises the Cas nuclease. In some embodiments, the recombinant plant virus used in the virus-mediated delivery is a negative strand RNA virus. In some embodiments, the recombinant plant virus used in the virus-mediated delivery has a segmented genome. In some embodiments, the recombinant plant virus used in the virus-mediated delivery further comprises an expression cassette comprising a reporter gene. In some embodiments, the reporter gene encodes a fluorescent reporter. In some embodiments, the recombinant plant virus is capable of cell-to-cell movement. In some embodiments, RNA encoding the gRNA and/or the Cas nuclease is delivered to the meristem cell of the soybean plant by transport from another plant tissue. In some embodiments, the Cas nuclease is delivered to the meristem cell of the soybean plant in a second viral vector comprising the Cas nuclease. In embodiments of the method, 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. In some embodiments, a viral vector may be delivered to a plant by transformation with Agrobacterium.

[0247] In another embodiment, a T-DNA vector is used to deliver at least one nucleic acid to plant cells. In some embodiments, a T-DNA binary vector is used. In some embodiments, a T-DNA superbinary vector system is used. In other embodiments, a T-DNA ternary vector system is used. In some embodiments, the T-DNA system further comprises an additional virulence gene cluster. In some embodiments, the T-DNA system further comprises an accessory plasmid or virulence helper plasmid. In some embodiments, the T-DNA vector is an Agrobacterium vector.

[0248] In some embodiments, 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.

[0249] In some embodiments, 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. B. Delivery of Genome Editing Reagents

[0250] Guide RNAs and Cas enzymes (collectively referred to here as “genome editing reagents”) that are aspects of the invention can be delivered to a plant cell using various techniques and agents. The polynucleotides, ribonucleoproteins, DNA expression systems, engineered systems, and vectors (collectively referred to here as “genome editing reagents”) that are aspects of the invention can be delivered to a plant cell using various techniques and agents. In some embodiments, the plant cell is a cell of a rootstock. In some embodiments, the plant cell is a cell of a leaf In some embodiments, the plant cell is a cell of a grafted scion. In some embodiments, the plant cell is a cell of a seed (including mature seed and immature seed). In some embodiments, the plant cell is a cell of a plant cutting. In some embodiments, the plant cell is a cell of a plant cell culture. In some embodiments, 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). In embodiments, 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. In an embodiment, 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 lysate, 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). For example, 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. For example, a plant cell or plant protoplast is abrased with powder and rubbed with the composition containing genome editing reagents. In embodiments, the genome editing reagentcontaining composition is delivered using negative or positive pressure, for example, using vacuum infiltration or application of hydrodynamic or fluid pressure. In embodiments, 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. In embodiments, the genome editing reagent-containing composition is provided to a plant cell or plant protoplast by bacterially mediated (e.g., Agrobacterium sp., Rhizobium sp., Sinorhiz.obium 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. Bacteria may be transformed by any method known in the art, including but not limited to electroporation. 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.

[0251] In some embodiments, 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. In embodiments, 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. In some embodiments, 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. In some embodiments, 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).

[0252] In some embodiments, a treatment employed in delivery of a genome editing reagent to a soybean plant, soybean plant cell, or soybean 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 soybean plant cell or soybean plant protoplast occurs), or heating or heat stress (exposure to temperatures above that at which normal growth of the soybean plant cell or soybean plant protoplast occurs), or treating at a combination of different temperatures. In embodiments, a specific thermal regime is carried out on a soybean plant cell or soybean plant protoplast, or on a soybean plant or soybean plant part from which a soybean plant cell or soybean plant protoplast is subsequently obtained or isolated, in one or more steps separate from the genome editing reagent delivery. In some embodiments, a specific thermal regime is carried out on a soybean plant cell, wherein the soybean plant cell is a cell of a rootstock. In some embodiments, a specific thermal regime is carried out on a soybean plant cell, wherein the soybean plant cell is a cell of a grafted scion. In some embodiments, a specific thermal regime is carried out on a soybean plant cell, wherein the soybean 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 soybean plant cell, wherein the soybean plant cell is a cell of a soybean plant cutting. In some embodiments, a specific thermal regime is carried out on a soybean plant cell, wherein the soybean plant cell is a cell of a soybean plant cell culture. In some embodiments, a specific thermal regime is carried out on a soybean plant cell, wherein the soybean plant cell is a cell of a soybean 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).

[0253] In some embodiments, a treatment employed in delivery of a genome editing reagent to a soybean plant, soybean plant cell, or soybean plant protoplast is carried out under a specific light regime, which can involve various photoperiods of light and/or dark stress on a soybean plant. In some embodiments, to kickstart BPMV viral infection upon viral vector delivery, soybean plants are additionally stressed by placing them in the dark at 20°C for 48h (24h prior and 24h after inoculation). In some embodiments, soybean plants are grown in a 16h day (22°C) 8h night (20°C) light regime and watered regularly beyond this stress period.

[0254] In some embodiments, 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. In embodiments, a genome editing reagent-containing composition further includes one or more one chemical, enzymatic, or physical agent for delivery. In some embodiments, the treated plant cell is a cell of a rootstock. In some embodiments, the treated plant cell is a cell of a grafted scion. In some embodiments, the Cas nuclease is delivered to the scion by transport from a grafted rootstock. In some embodiments, the methods provided herein further comprises transforming the rootstock with a nucleic acid encoding the Cas nuclease prior to grafting. In some embodiments, the scion and the rootstock are the same plant species. In some embodiments, the scion and the rootstock are different plant species. In some embodiments, the rootstock is canola, alfalfa, corn, oat, sorghum, sugarcane banana, or wheat. In some embodiments, the treated plant cell or plant protoplast is not a soybean. In some embodiments, the treated plant cell is a cell of a seed (including mature seed and immature seed). In some embodiments, 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). 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. In embodiments, 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). In non-limiting examples, 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. Examples of 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.

[0255] In some embodiments, a whole soybean plant or soybean plant part or soybean seed, or an isolated soybean plant cell or soybean plant protoplast, or the soybean plant or plant part from which a soybean plant cell or soybean 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. In embodiments, a genome editing reagent-containing composition further includes one or more one chemical, enzymatic, or physical agent for delivery. In some embodiments, the treated soybean plant cell is a cell of a rootstock. In some embodiments, the treated soybean plant cell is a cell of a grafted scion. In some embodiments, the Cas nuclease is delivered to the scion by transport from a grafted rootstock. In some embodiments, the methods provided herein further comprises transforming the rootstock with a nucleic acid encoding the Cas nuclease prior to grafting. In some embodiments, the scion and the rootstock are the same plant species. In some embodiments, the scion and the rootstock are different plant species. In some embodiments, the rootstock is canola, alfalfa, corn, oat, sorghum, sugarcane banana, or wheat. In some embodiments, the treated plant cell or plant protoplast is not a soybean. In some embodiments, the treated soybean plant cell is a cell of a soybean seed (including mature seed and immature seed). In some embodiments, the treated soybean plant cell is a cell of a soybean plant cutting. In some embodiments, the treated soybean plant cell is a cell of a soybean plant cell culture. In some embodiments, the treated soybean plant cell is a cell of a soybean 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). 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. In embodiments, 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). In non-limiting examples, 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 cellpenetrating peptide. Examples of 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.

[0256] The polynucleotides, ribonucleoproteins, DNA expression systems, engineered systems, vectors, guide RNAs, and Cas enzymes (collectively referred to here as “genome editing reagents”) that are aspects of the invention can be delivered to a plant cell using various techniques and agents. In some embodiments, the plant cell is a cell of a rootstock. In some embodiments, the plant cell is a cell of a leaf. In some embodiments, the plant cell is a cell of a grafted scion. In some embodiments, the plant cell is a cell of a seed (including mature seed and immature seed). In some embodiments, the plant cell is a cell of a plant cutting. In some embodiments, the plant cell is a cell of a plant cell culture. In some embodiments, 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). In some embodiments, 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. In an embodiment, 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 sap, 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 soybean plant cell or soybean 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 genome editing reagentcontaining composition in the form of a lysate, 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 soybean plant cell or soybean 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). For example, a soybean plant cell or soybean plant protoplast is soaked in a liquid genome editing reagent-containing composition, whereby the genome editing reagent is delivered to the soybean plant cell or soybean plant protoplast. In embodiments, the genome editing reagentcontaining composition is delivered using negative or positive pressure, for example, using vacuum infiltration or application of hydrodynamic or fluid pressure. In embodiments, the genome editing reagent-containing composition is introduced into a soybean plant cell or soybean 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. In another example of delivery to the soybean plant cell or soybean plant protoplast, a soybean plant cell or soybean plant protoplast is abrased with powder and rubbed with the composition containing genome editing reagents. Other techniques useful for delivering the genome editing reagentcontaining 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. In embodiments, the genome editing reagent-containing composition is provided to a soybean plant cell or soybean plant protoplast by bacterially mediated (e.g., Agrobacterium sp., Rhizobium sp., Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., Phyllobacterium sp.) transfection of the soybean plant cell or soybean plant protoplast with a polynucleotide encoding the gRNA; see, e.g., Broothaerts et al. Nature 2005, 433: 629-633. Bacteria may be transformed by any method known in the art, including but not limited to electroporation. Any of these techniques or a combination thereof are alternatively employed on the soybean plant part or tissue or intact soybean plant (or seed) from which a soybean plant cell or soybean plant protoplast is optionally subsequently obtained or isolated; in embodiments, the genome editing reagent-containing composition is delivered in a separate step after the soybean plant cell or soybean plant protoplast has been obtained or isolated.

[0257] In some embodiments, a treatment employed in delivery of a genome editing reagent to a soybean plant, soybean plant cell, or soybean 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 soybean plant cell or soybean plant protoplast occurs), or heating or heat stress (exposure to temperatures above that at which normal growth of the soybean plant cell or soybean plant protoplast occurs), or treating at a combination of different temperatures. In embodiments, a specific thermal regime is carried out on a soybean plant cell or soybean plant protoplast, or on a soybean plant or soybean plant part from which a soybean plant cell or soybean plant protoplast is subsequently obtained or isolated, in one or more steps separate from the genome editing reagent delivery. In some embodiments, a specific thermal regime is carried out on a soybean plant cell, wherein the soybean plant cell is a cell of a rootstock. In some embodiments, a specific thermal regime is carried out on a soybean plant cell, wherein the soybean plant cell is a cell of a grafted scion. In some embodiments, a specific thermal regime is carried out on a soybean plant cell, wherein the soybean 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 soybean plant cell, wherein the soybean plant cell is a cell of a soybean plant cutting. In some embodiments, a specific thermal regime is carried out on a soybean plant cell, wherein the soybean plant cell is a cell of a soybean plant cell culture. In some embodiments, a specific thermal regime is carried out on a soybean plant cell, wherein the soybean plant cell is a cell of a soybean 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).

[0258] In some embodiments, a treatment employed in delivery of a genome editing reagent to a soybean plant, soybean plant cell, or soybean plant protoplast is carried out under a specific light regime, which can involve various photoperiods of light and/or dark stress on a soybean plant. In some embodiments, to kickstart BPMV viral infection upon viral vector delivery, soybean plants are additionally stressed by placing them in the dark at 20°C for 48h (24h prior and 24h after inoculation). In some embodiments, soybean plants are grown in a 16h day (22°C) 8h night (20°C) light regime and watered regularly beyond this stress period.

[0259] In some embodiments, a whole soybean plant or plant part or seed, or an isolated soybean plant cell or soybean plant protoplast, or the soybean plant or plant part from which a soybean plant cell or soybean 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. In embodiments, a genome editing reagent-containing composition further includes one or more one chemical, enzymatic, or physical agent for delivery. In some embodiments, the treated soybean plant cell is a cell of a rootstock. In some embodiments, the treated soybean plant cell is a cell of a grafted scion.

[0260] Compositions comprising: (i) RNA molecules comprising an MTS linked to a Cas nuclease and/or guide RNA(s); (ii) nucleic acids encoding RNA guided nucleases; (iii) a BPMV viral vector linked to a Cas nuclease and/or guide RNA(s); and/or (iv) donor DNA templates can further comprise components that include:

(a) solvents (e.g., water, dimethylsulfoxide, dimethylformamide, acetonitrile, N-pyrrolidine, pyridine, hexamethylphosphor amide, alcohols, alkanes, alkenes, dioxanes, polyethylene glycol, and other solvents miscible or emulsifiable with water or that will dissolve phosphonucleotides in non-aqueous systems);

(b) fluorocarbons (e.g., perfluorodecalin, perfluoromethyldecalin);

(c) glycols or polyols (e.g., propylene glycol, polyethylene glycol);

(d) 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; tallow amine; 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. Louis, MO); long chain alcohols; 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. 5905-50073-AA, Momentive Performance Materials, Inc., Albany, N.Y.); specific examples of useful 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;

(e) lipids, lipoproteins, lipopolysaccharides;

(f) acids, bases, caustic agents; buffers;

(g) peptides, proteins, or enzymes (e.g., cellulase, pectolyase, maceroenzyme, pectinase), including cellpenetrating or pore-forming peptides (e. g., (B0100)2K8, Genscript; poly-lysine, 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. g., www[dot]lifetein[dot]com/Cell_Penetrating_Peptides[dot]html and Jarver Mol. Therapy-Nucleic Acids 2012, 1: e27,l - 17); octa-arginine or nona-arginine; poly-homoarginine (see Unnamalai et al. FEBS Letters 2004, 566: 307 - 310); see also 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)

(h) RNase inhibitors;

(i) 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); (j) dendrimers (see, e. g., US Patent Application Publication 2011/0093982, incorporated herein by reference in its entirety);

(k) counter-ions, amines or polyamines (e. g., spermine, spermidine, putrescine), osmolytes, buffers, and salts (e. g., calcium phosphate, ammonium phosphate);

(l) polynucleotides (e. g., non-specific double-stranded DNA, salmon sperm DNA);

(m) 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);

(n) antibiotics, including non-specific DNA double- strand-break-inducing agents (e. g., phleomycin, bleomycin, talisomycin);

(o) antioxidants (e. g., glutathione, dithiothreitol, ascorbate); and/or

(p) chelating agents (e. g., EDTA, EGTA).

[0261] In embodiments, the chemical agent is provided simultaneously with the genome editing reagent. In embodiments, 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). In embodiments, 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.

[0262] In embodiments, 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 Magnetotransfection™ agent, OZ Biosciences, San Diego, Calif.), abrasive or scarifying agents, needles or microneedles, matrices, and grids. In embodiments, 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-ethylenedioxy thiophene) 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). In embodiments, 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). In various embodiments, such particulates and nanoparticles are neutral, or carry a positive charge, or carry a negative charge. 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. Other embodiments of useful particulates are 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. Specific, non-limiting embodiments of 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. (2016) Nano Lett., 16: 1161-1172; Giraldo et al. (2014) Nature Materials, 13:400-409; Shen et al. (2012) Theranostics, 2:283-294; Kim et al. (2011) Bioconjugate Chem., 22:2558-2567; Wang et al. (2010) J. Am. Chem. Soc. Comm., 132:9274-9276; Zhao et al. (2016) Nanoscale Res. Lett., 11:195-203; and Choi et al. (2016) J. Controlled Release, 235:222-235. See also, for example, the various types of particles and nanoparticles, their preparation, and methods for their use, e.g., in delivering polynucleotides and polypeptides to cells, disclosed in U.S. Patent Application Publications 2010/0311168, 2012/0023619, 2012/0244569, 2013/0145488, 2013/0185823, 2014/0096284, 2015/0040268, 2015/0047074, and 2015/0208663, all of which are incorporated herein by reference in their entirety.

[0263] In embodiments, 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). In some embodiments, 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 Cas nuclease). In some embodiments, the treated plant cell is a cell of a rootstock. In some embodiments, the treated plant cell is a cell of a grafted scion. In some embodiments, the treated plant cell is a cell of a seed (including mature seed and immature seed). In some embodiments, 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). In embodiments, 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. In embodiments, the genome editing reagent is applied to adjacent or distal cells or tissues and is transported (e.g., through the vascular system or by cell-to-cell movement) to the meristem from which plant cells or plant protoplasts are subsequently isolated. In embodiments, 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. In embodiments, 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. In embodiments, a genome editing reagent-containing 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. In embodiments 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.

[0264] Compositions comprising: (i) DNA molecules comprising a BPMV sequence linked to a Cas nuclease and guide RNA(s); (ii) nucleic acids encoding RNA guided nucleases; and/or (iii) donor DNA templates can be delivered to the plant and/or meristem cells of the plant by any method of delivery. Compositions comprising: (i) RNA or DNA molecules comprising an MTS linked to a Cas nuclease and/or guide RNA(s), or a viral vector comprising the Cas nuclease and/or the guide RNA(s); (ii) nucleic acids encoding RNA guided nucleases; 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 cell-penetrating peptides. In some embodiments, 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. In some embodiments, 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).

[0265] In certain embodiments, plants are contacted either simultaneously or sequentially with one, two, three or more BPMV vectors carrying a guide RNA. In some embodiments, the guide RNA is linked to a BPMV sequence. In certain embodiments, 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 RNA fused to an MTS. In certain embodiments, plants are contacted either simultaneously or sequentially with one, two, three or more viral vectors carrying a guide RNA. In some embodiments, the guide RNA is linked to a viral vector. In some embodiments, the guide RNA is linked to a recombinant plant virus. In some embodiments, the guide RNA is linked to a BPMV sequence. In some embodiments, the sequence of BPMV comprises the BPMV genomic segment RNA2. In some embodiments, the viral vector carrying the guide RNA is present in a composition that contacts a soybean plant. In some embodiments, the BPMV vector carrying the guide RNA is present in a composition that contacts a soybean plant. In some embodiments, the composition contacts a soybean seed (including mature seed and immature seed). In some embodiments, the composition contacts a soybean plant cutting. In some embodiments, the composition contacts a soybean plant cell culture.

[0266] In some embodiments, the methods herein further comprise infecting the soybean plant with a plurality of viral vectors, wherein each viral vector comprises one or more gRNA and/or the Cas nuclease. In some embodiments, the plurality of viral vectors co-infect the soybean plant simultaneously or infect the soybean plant in more than one round of infection. In some embodiments, the composition contacts a rootstock. In some embodiments, the composition contacts a grafted scion. In some embodiments, the composition contacts a seed (including mature seed and immature seed). In some embodiments, the composition contacts a plant cutting. In some embodiments, the composition contacts a soybean plant cutting. In some embodiments, the composition contacts a plant cell culture. In some embodiments, 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). In certain embodiments, 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 an RNA encoding a Cas nuclease fused to an MTS. In some embodiments, the composition contacts a plant cell culture. In some embodiments, the composition contacts a soybean plant cell culture. In some embodiments, the composition contacts a soybean 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, root, flowers, fruits, shoots, and explants). In certain embodiments, plants are contacted either simultaneously or sequentially with one, two, three or more viral vectors in one or more compositions where at least one of the viral vectors or viral vector systems comprises an RNA encoding a Cas nuclease, optionally with the Cas nuclease fused to an MTS. In certain embodiments, one of the RNA molecules comprises a guide RNA fused to an MTS and a second RNA molecule comprises RNA encoding an RNA guided Cas nuclease and optionally an MTS, where the RNA guided Cas nuclease can process the RNA comprising the guide RNA to release a functional guide RNA. In certain embodiments, one of the RNA molecules comprises at least one guide RNA fused to an MTS and a second RNA 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). In certain embodiments, 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). In certain embodiments, 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. In certain embodiments, the guide RNA(s) delivered by the first viral vector is distinct from the guide RNA(s) delivered by the second viral vector. Such distinct gRNAs provided by the first viral vector can provide for genome editing at one or more first genomic sites in a meristem cell while the distinct gRNAs provided by the second viral vector can provide for genome editing at one or more second genomic sites in a meristem cell. Such contacting the plant with viral vectors 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 viral vector 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. In certain embodiments, the guide RNA(s) delivered by the first BPMV vector is distinct from the guide RNA(s) delivered by the second BPMV vector. Such distinct gRNAs provided by the first BPMV vector can provide for genome editing at one or more first genomic sites in a meristem cell while the distinct gRNAs provided by the second BPMV vector can provide for genome editing at one or more second genomic sites in a meristem cell. Such contacting the plant with BPMV vectors 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 BPMV vector 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. Without seeking to be limited by theory, it is believed that cutting chromosomes at multiple location simultaneously is cytotoxic and that such cytotoxicity can be mitigated by delivering a limited number of guide RNAs at different times (e.g., about 6, 12, 18, or 20 to about 24, 30, or 36 hours apart). In certain embodiments, 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. Without seeking to be limited by theory, it is believed that cutting chromosomes at multiple location simultaneously is cytotoxic and that such cytotoxicity can be mitigated by delivering a limited number of guide RNAs at different times (e.g., about 6, 12, 18, or 20 to about 24, 30, or 36 hours apart). In certain embodiments, a soybean plant can be contacted by one or more RNA molecules or viral vectors 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, optionally 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.

[0267] In certain embodiments, soybean plants are contacted either simultaneously or sequentially with one, two, three or more BPMV vectors or vector systems in one or more compositions where at least one of the BPMV vectors or vector systems comprises an RNA encoding a Cas nuclease fused to the BPMV vector. In certain embodiments, one of the BPMV vectors carries a guide RNA fused to a BPMV-RNA2 genomic segment and a second BPMV vector carries an RNA guided Cas nuclease. In certain embodiments, one of the BPMV vectors carries a guide RNA fused to a BPMV-RNA1 genomic segment and a second BPMV vector carries an RNA guided Cas nuclease. In some embodiments, the RNA guided Cas nuclease can process the BPMV vector carrying the guide RNA to release a functional guide RNA. In certain embodiments, one of the BPMV vectors carries at least one guide RNA fused to a BPMV vector and a second BPMV vector comprises an RNA guided nuclease, and optionally a Cas enzyme. In certain embodiments, guide RNAs are flanked by or comprise processing elements (e.g., DRs) which are processed by different RNA-guided nucleases. In certain embodiments, the guide RNA(s) delivered by the first BPMV vector is distinct from the guide RNA(s) delivered by the second BPMV vector. Such distinct gRNAs provided by the first BPMV vector can provide for genome editing at one or more first genomic sites in a meristem cell while the distinct gRNAs provided by the second BPMV vector can provide for genome editing at one or more second genomic sites in a meristem cell. Such contacting the soybean plant with BPMV vectors 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 soybean plant with the second BPMV vector 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. Without seeking to be limited by theory, it is believed that cutting chromosomes at multiple location simultaneously is cytotoxic and that such cytotoxicity can be mitigated by delivering a limited number of guide RNAs at different times (e.g., about 6, 12, 18, or 20 to about 24, 30, or 36 hours apart). In certain embodiments, a soybean plant can be contacted by one or more BPMV vectors that comprise at least one gRNA fused to a BPMV-RNA2 genomic segment, optionally along with a Cas nuclease, permitted a sufficient period of time to accumulate the BPMV vector 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 BPMV vectors that comprise at least one different gRNA fused to a BPMV-RNA2 genomic segment, optionally along with an RNA encoding an RNA guided Cas nuclease, where the RNA guided Cas nuclease can process the guide RNA(s) to release a functional guide RNA and/or effect a desired genomic edit with the gRNA in the meristem cells. In certain embodiments, a soybean plant can be contacted by one or more BPMV vectors that comprise at least one gRNA fused to a BPMV-RNA1 genomic segment, optionally along with a Cas nuclease, permitted a sufficient period of time to accumulate the BPMV vector 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 BPMV vectors that comprise at least one different gRNA fused to a BPMV-RNA1 genomic segment, optionally along with an RNA encoding an RNA guided Cas nuclease, where the RNA guided Cas nuclease can process the guide RNA(s) to release a functional guide RNA and/or effect a desired genomic edit with the gRNA in the meristem cells.

[0268] Guide RNAs 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 linked to an MTS. Guide RNAs can be provided to a plant part and transported to a meristem cell by a variety of methods that include stable expression with an integrated transgene or transient expression such as by introducing a viral vector that carries the gRNA. In some embodiments, the viral vector is a BPMV vector. In certain embodiments, the gRNA is predominantly localized in meristem tissue of the plant. Delivery of viral vector systems encoding the gRNA(s) or DNA(s)/RNA(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. Delivery of 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. In some embodiments, the gRNA(s) are delivered to a rootstock. In some embodiments, the gRNA(s) are delivered to a grafted scion. In some embodiments, the gRNA(s) are delivered to a seed (including mature seed and immature seed). In some embodiments, the gRNA(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 gRNA(s) are delivered to a plant cell culture. In some embodiments, the gRNA(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).

[0269] In some embodiments, a guide RNA for the Cas nuclease is applied to a leaf, a shoot, a stem, and/or meristem of the plant. In some embodiments, a composition comprising the guide RNA for the Cas nuclease is applied to a leaf, a shoot, a stem, and/or meristem of the plant. In some embodiments, the composition comprising the guide RNA comprises a nuclease inhibitor. In some embodiments, the composition comprising the guide RNA comprises an RNase inhibitor. In some embodiments, a guide RNA for the Cas nuclease is applied to a leaf, a shoot, a stem, a cell culture, any vegetative tissue, and/or meristem-associated cells of the plant. In some embodiments, a composition comprising the guide RNA for the Cas nuclease is applied to a leaf, a shoot, a stem, a cell culture, any vegetative tissue, and/or meristem-associated cells of the plant. In some embodiments, the composition comprising the guide RNA comprises a nuclease inhibitor. In some embodiments, the composition comprising the guide RNA comprises an RNase inhibitor.

In some embodiments, a guide RNA for the Cas nuclease is applied to a leaf, a shoot, a stem, and/or meristem of the plant. In some embodiments, a composition comprising the guide RNA for the Cas nuclease is applied to a leaf, a shoot, a stem, a cell culture, any vegetative tissue, and/or meristem of the plant.

[0270] In some embodiments, delivery of the guide RNA comprises spraying a composition comprising the guide RNA onto the leaves, shoot, stem, and/or meristem. In some embodiments, the composition comprising the guide RNA comprises a surfactant. In some embodiments, the composition comprising the guide RNA comprises glass beads coated with the guide RNA.

[0271] In some embodiments, delivery of the guide RNA comprises rubbing a composition comprising the guide RNA onto the leaves, shoot, stem, and/or meristem. In some embodiments, delivery of the guide RNA comprises rubbing a composition comprising a viral vector comprising the guide RNA onto the leaves, shoot, stem, and/or meristem.

[0272] In some embodiments, delivery of the guide RNA comprises injecting a composition comprising the guide RNA into the stem. In some embodiments, delivery of the guide RNA comprises injecting a composition comprising a viral vector comprising the guide RNA into the stem. In some embodiments, delivery of the guide RNA comprises injecting a composition comprising BPMV vector carrying the guide RNA into the stem.

[0273] In some embodiments, delivery of the guide RNA comprises leaf infiltration of a composition comprising the guide RNA into a leaf. In some embodiments, delivery of the guide RNA comprises leaf infiltration of a composition comprising the BPMV vector carrying the guide RNA into a leaf. In some embodiments, the leaf infiltration comprises forced infiltration using a needle-less syringe or vacuum pump. In some embodiments, delivery of the guide RNA comprises leaf infiltration of a composition comprising a viral vector comprising the guide RNA into a leaf. In some embodiments, the leaf infiltration comprises forced infiltration using a needle-less syringe or vacuum pump.

[0274] In some embodiments, delivery of a guide RNA for the Cas nuclease comprises biolistic transformation of nucleic acid encoding the guide RNA into the leaf, shoot, stem, and/or meristem. In some embodiments, the biolistic transformation comprises transformation of circular DNA encoding the guide RNA.

[0275] In other embodiments, a guide RNA for the Cas nuclease is delivered to the roots of the plant. In some embodiments, a composition comprising the guide RNA for the Cas nuclease is applied to the roots. In some embodiments, the composition comprising the guide RNA comprises a nuclease inhibitor. In some embodiments, the composition comprising the guide RNA comprises an RNase inhibitor. [0276] In some embodiments, the guide RNA is delivered to the plant root by incubating the root with a composition comprising the guide RNA.

[0277] In some embodiments, a guide RNA for the Cas nuclease is delivered to the plant root by Agrobacterium rhizogenes transformation.

[0278] RNA guided nucleases 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 RNA guided nuclease or an RNA that encodes the RNA guided nuclease that is linked to an MTS. In certain embodiments, an active form of the RNA guided nuclease is predominantly localized in root tissue of the plant. In certain embodiments, the RNA guided nuclease can be 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. RNA guided nucleases, including Cas nucleases, can be provided to at least the meristem cell by a variety of methods that include stable expression, such as with an integrated transgene, expression from a viral vector, or transient expression such as by introducing an RNA that encodes the RNA guided nuclease or an RNA that encodes the RNA guided nuclease that is linked or fused to a viral vector and/or to an MTS. In certain embodiments, an active form of the RNA guided nuclease is predominantly localized in root tissue of the plant. In certain embodiments, the RNA guided nuclease can be 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.

[0279] RNA guided nucleases, including Cas enzymes, can be provided to a plant part and transported to a meristem cell by a variety of methods that include stable expression with an integrated transgene or transient expression such as by introducing a viral vector that carries the gRNA.

[0280] RNA guided nucleases, including Cas enzymes, can be provided to at least the meristem cell by a variety of methods that include stable expression, such as with an integrated transgene, or transient expression such as by introducing an RNA that encodes the RNA guided nuclease or an RNA that encodes the RNA guided nuclease that is linked to a BPMV genomic segment.

[0281] In some embodiments, a plant expressing transgenically a Cas polypeptide may be genomically edited by delivery of a second RNA containing only guide RNAs suitable for the transgenically expressed Cas polypeptide. In some embodiments, a plant expressing transgenically a Cas polypeptide may be genomically edited by delivery of a second viral vector or viral vector system containing only guide RNAs suitable for the transgenically expressed Cas polypeptide. In some embodiments, a plant expressing transgenically a Cas polypeptide may be genomically edited by delivery of a BPMV vector system containing only guide RNAs suitable for the transgenically expressed Cas polypeptide. In some embodiments, a plant expressing transgenically a Cas polypeptide may be genomically edited by delivery of a second viral vector or viral vector system containing only guide RNAs suitable for the transgenically expressed Cas polypeptide.

[0282] The 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.

[0283] 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. For example, one RNA may comprise a Cas nuclease while another may comprise a corresponding guide or guide array. In addition, a substantially purified RNA composition may comprise other added components, such as a pH buffer, salt, surfactants, and/or RNase inhibitors.

[0284] 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.

[0285] The 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. In certain embodiments, 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). In certain embodiments, 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 (Corn 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”).

[0286] The viral vectors 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 viral vectors, RNA molecules, or compositions comprising the RNA molecules or viral vectors, to floral meristems, especially at early stages of differentiation. In certain embodiments, a soybean plant is contacted at the vegetative stage with a composition comprising the RNA molecules or viral 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). In certain embodiments, 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 (Corn 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”).

[0287] The viral vectors 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 viral vectors, or compositions comprising the viral vectors, to floral meristems, especially at early stages of differentiation. In certain embodiments, a soybean plant is contacted at the vegetative stage with a composition comprising the viral 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).

EXAMPLES

Example 1: Bean Pod Mottle Virus (BPMV) Constructs

[0288] Bean pod mottle virus (BPMV, genus Comovirus) has a bipartite positive RNA genome consisting of RNA1 (GenBank: NC003496) and RNA2 (GenBank: NC003495). BPMV-RNA1 and BPMV-RNA2 are known to be expressed as a single polyprotein precursor which subsequently undergoes proteolysis to yield mature viral gene products. To test virus-induced gene editing (VIGE) in soybean, BPMV was engineered to carry three different CRISPR guide RNAs (gRNAs) targeting sites within a soybean gene encoding the enzyme phytoene desaturase (PDS).

[0289] Vector constructs containing cDNA copies of BMPV RNA1 (pIN4100) and RNA2 (pIN4102) were provided by Steve Whitham (Iowa State University) and guide sequences (crRNA) were introduced at the 3 ’-end of RNA2 using standard cloning procedures (Zhang et al. Development and use of an efficient DNA-based viral gene silencing vector for soybean. Mol Plant Microbe Interact. 2009. 22 123-131), resulting in the following constructs:

[0290] pIN4656 contained crRNA_l_PDS specific for phytoene desaturase genes GmPDSl l (Glyma.l lG253000; SEQ ID NO: 63) and GmPDS18 (Glyma.l8G003900; SEQ ID NO: 64) from soybean. crRNA_l_PDS was composed of target sequence (spacer: GTAAGAAGCTCTTCACCGTTCCA; SEQ ID NO: 69) flanked by two direct repeats (DR-spacer-DR; also referred to as DR-DR configuration or DR-DR design).

[0291] pIN4917 contained crRNA_2_PDS specific for phytoene desaturase genes GmPDSl l (Glyma.l 1G253000; SEQ ID NO: 63 in table below) and GmPDS18 (Glyma.l8GOO39OO; SEQ ID NO: 64 in table below) from soybean. crRNA_2_PDS was composed of target sequence (spacer: GTAAGAAGCTCTTCACCGTTCCA; SEQ ID NO: 69) flanked by two direct repeats, preceded by a caged, truncated, pre-tRNA-like structure (catRNA; Zhang et al. 2018). [0292] pIN4652 contained crRNA_3_PDS (CR_1991_GmPDS_Ex7) specific for phytoene desaturase genes GmPDSl l (Glyma.l lG253000; SEQ ID NO: 63) and GmPDS18 (Glyma.l8G003900; SEQ ID NO: 64) from soybean. crRNA_3_PDS was composed of target sequence (spacer: GTAAGAAGCTCTTCACCGTTCCA; SEQ ID NO: 69) preceded by one direct repeat, and flanked at both ends by two (HH and HDV) self-cleaving ribozyme sequences (HH-DR-spacer-HDV ; referred to as ribozyme configuration).

Example 2 - Stable Transformation of Soybean Plants with Binary Vectors for the overexpression of CasS (any suitable Cas) nuclease

[0293] Stable transgenic Glycine max soybean plants “NINF1170” (T4 generation) expressing Cas protein were produced by Agrobacterium-mediated genetic transformation (EHA 105 strain, plasmid construct pIN1722) using the cotyledonary-node method.

[0294] The Agrobacterium transformation process included the following steps: explants were obtained after seed sterilization; explants were infected with the Agrobacterium suspension liquid and co-cultivated on the co-cultivation medium (CCM); the Agrobacterium-mfected explants were transferred to shoot induction medium (SIM); the explants were then transferred to shoot elongation medium (SEM); the elongated shoots were placed in rooting medium; and the seedlings were then transferred to pots to grown to maturity.

Example 3: Mechanical inoculation of soybean plants with BPMV viral constructs for production of infectious lysate

[0295] The unifoliate leaves from 12-day old Glycine max wild type soybean plants “NINF1170” were inoculated with BPMV following a direct DNA mechanical inoculation procedure (Pflieger, S. et al. Bean Pod Mottle Virus (BPMV) Viral Inoculation Procedure in Common Bean (Phaseolus vulgaris L.). BIO-PROTOCOL. 2015. 5. 10.21769/BioProtoc[dot] 1524).

[0296] An infectious plasmid DNA mixture harbouring the two constructs, BPMV-RNA1 (pIN4100) and the guide-containing BPMV-RNA2 (pIN4102, or modified guide constructs as listed in Example 1), was prepared for each unifoliate leaf that was inoculated. Per leaf, roughly 5000 ng of plasmid DNA of each construct was mixed into a 10 mM Phosphate buffer (pH 7.0), to a final volume of 20 pL.

[0297] Carborundum powder was dusted on the adaxial side of both unifoliate leaves, the infectious plasmid DNA mixture was pipetted onto the leaf, and the entire surface of the leaf was gently rubbed with this mixture. After inoculation, the leaves were rinsed with demineralized water to rinse away the surplus of carborundum powder.

[0298] To kickstart the viral infection, the plants were additionally stressed by placing them in the dark at 20°C for 48h (24h prior and 24h after inoculation). The plants were grown in a 16h day (22°C) 8h night (20°C) light regime and watered regularly. [0299] Once fully developed (V3/V4 growth stage), the third trifoliate leaf was sampled for RNA isolation by collecting six leaf discs of 6 mm diameter. After cDNA synthesis, molecular analysis was performed for confirming viral presence (via qPCR for coat protein). The presence of the intact guide RNA sequences in the same plants was confirmed by Sanger sequencing of systemically infected leaves. The virus clones carrying the PDS guide RNA was used to further inoculate plants (Example 4).

[0300] Infection phenotype was evaluated by comparison to untreated soybean plants (MOCK inoculated/negative controls; FIG. 1). Plants inoculated with wild type BPMV constructs developed leaf mottling typical for BPMV infection.

[0301] The fourth and fifth leaves from selected plants showing high viral load and confirmed guide sequences were collected as viral inoculum. When not used directly, infected leaf material was lyophilized and kept at -20°C for later use.

Example 4: Mechanical inoculation of Cas expressing soybean plants with BPMV infected leaf lysate

[0302] The unifoliate leaves from 12-day old Glycine max Cas-expressing plants were inoculated with a potent viral inoculum (obtained in Example 3). This potent viral inoculum was prepared by grinding selected leaves (fresh or lyophilized) using pestle and mortar in presence of 10 mM sodium phosphate buffer, pH 7.0.

[0303] Carborundum powder (superfine 600 grit; Thermo Scientific Chemicals) was dusted on the adaxial side of both unifoliate leaves, and a piece of Miracloth™ (Merck Chemicals N. V. ; Millipore) 4 cm*4 cm was drenched in the potent viral inoculum until it was fully soaked. The entire surface of each unifoliate leaf was individually, gently rubbed. The leaves were then left to dry (about 5 minutes), after which they were rinsed with distilled water.

[0304] To kickstart the viral infection the plants were additionally stressed by placing them in the dark at 20°C for 48h (24h prior and 24h after inoculation). The plants were grown in a 16h day (22°C) 8h night (20°C) regime and watered regularly.

[0305] Once fully developed (V3/V4 growth stage), the third trifoliate leaf was sampled for genomic DNA and total RNA isolation (by collecting six leaf discs of 6 mm diameter). The samples were snap- frozen in liquid nitrogen and stored at -80°C for later processing and subsequent analysis.

[0306] DNA and RNA were isolated in parallel from the same samples using Direct-zol™-96 MagBead RNA (Zymo Research) according to manufacturer’s instructions, with the exception that TRIzol® was replaced with Buffer RLT (Qiagen, Venlo, the Netherlands) containing 10 mM DTT. The same lysate was used to isolate genomic DNA using CleanNGS magnetic beads (CleanNS; The Netherlands) according to standard protocols.

[0307] Isolated RNA was used for first strand cDNA synthesis using SuperScript™ IV VILO™ Master Mix (Thermo Fisher Scientific), and further analysis was performed for confirming viral presence (qPCR for coat protein) and guide sequence integrity (Sanger sequencing). [0308] The presence of the intact guide RNA sequences in planta was confirmed by Sanger sequencing, and amplicon sequencing was performed to identify editing at the target PDS site (Example 5).

[0309] The plants were also checked for phenotype. Almost all plants inoculated with viral RNA developed virus symptoms; additionally, most plants inoculated with BPMV carrying a PDS crRNA showed clear bleached phenotype characteristic for a non-functional PDS gene (FIG. 1).

[0310] Systemic spread of the virus continued throughout plant development. Additionally, for edited plants, the PDS phenotype continuously spread across all developing stages, including the seed pods (FIG. 1, right).

Example 5: Genotyping of BPMV -induced PDS knockout plants using amplicon-sequencing

[0311] Cas-transgenic soybean plants (from Example 4) inoculated with BPMV-RNA1 (pIN4100) together with RNA2-crRNA_l_PDS (pIN4656), RNA2-crRNA_2_PDS (pIN4917), RNA2- crRNA_3_PDS (pIN4652), wild type-BPMV-RNA2 (pIN4102) and mock inoculation were selected for deep sequencing analysis. Reference sequences of phytoene desaturase genes from soybean are listed in Table 4.

[0312] Soybean has two genes coding for PDS: PDS 11 (Glyma.l lg253000; SEQ ID NO: 63) and PDS 18 (Glyma.l8g003900; SEQ ID NO: 64). Reference sequence used for deep sequencing analysis consists of 215 nucleotides. PDS11 and PDS18 amplicons have 3 mismatches in nucleotide positions 48, 51 and 195. crRNA target sequence (nucleotide position 146-169:

GTAAGAAGCTCTTCACCGTTCCA, SEQ ID NO: 69) is identical for both PDS11 and PDS18. Sequencing is shown in FIG. 2.

[0313] Genomic DNA from 21 dpi (days post-infection) plant material was used for first-round PCR with Phusion Flash High-Fidelity PCR Master Mix (Thermo Fisher Scientific, Waltham, Mass., USA) introducing adaptor sequences and wobble bases needed for second PCR and sequencing. At this stage, amplicon sequencing was chosen for the two PDS genes combined, so two primers were used which can amplify both PDS genes indiscriminately: PDS_ex7 fwd (CTATAGGAGAAACATGGTTCTA; SEQ ID NO: 80) and PDS_ex7_rev (GTTGCAAACACATAAGCATC; (SEQ ID NO: 81)). This oligo pair amplified fragments of both soybean PDS 11 and PDS 18 genes.

[0314] PCR was carried out with 35 cycles, from which the product was loaded on a 2% agarose gel (5pl of each sample was mixed with Ipl purple loading dye, loaded together with a Ikb plus ladder (NEB) and run for 45 min at 100V), for confirming successful amplification. Remaining PCR product was cleaned with CleanNGS magnetic beads (CleanNA, The Netherlands) and quantified using Invitrogen™ Quant-iT™ IX dsDNA Assay Kits broad range (Invitrogen, Thermo Fisher Scientific).

[0315] Pooled amplicon sequencing of PDS target region, using the Illumina® MiSeq™ platform, was used to assess the editing efficiencies of the target gene and identify edited (frameshift) allele variants. During the first-round PCR, genomic regions containing target site were amplified, using genespecific primers with overhang adaptor and wobble/degenerated sequences (described in the paragraph above), for library preparation using the Tecan Fluent780 liquid handler and KAPA HiFi plus dNTPs kit (Roche, Basel, Switzerland). Equimolar amounts of all amplicons were mixed in a single tube and then used to prepare a single-indexed MiSeq™ library during the second-round PCR, by means of a pair of Illumina® DNA Prep primers. The amplicon library was sequenced by using MiSeq™ Reagent Kit v2 (Illumina® MS-102-2003, California, USA). Adequate numbers of sequence reads were simultaneously obtained to estimate the mutation rate for the different experimental conditions in a single-indexed library.

[0316] Sequencing data were subjected to InDei analysis, meaning the reads were analyzed for insertions and deletions, and mapped to the respective non-edited amplicon reference. The reads for every sample were trimmed (cutting of adaptor, wobble, and primer sequences) and aligned to the 225 nt of the PDS reference sequence (PDS11 and PDS18 combined). Identical sequences were summarized in one cluster with the respective size (number of reads edited compared to total reads mapped to target). The cluster with the biggest size corresponding to few nucleotide deletions in the genomic DNA target region is named as variant 1, and the second cluster as variant largest cluster as variant2, and so on.

[0317] Most edited variants showed deletions around the crRNA target region from one to 14 nucleotides (up to 26 nt in a few cases) but no nucleotide insertion. Summarized results across replicates are shown in Table 3.

Table 3. Overview table summarizing deep sequencing results. Values shown depict frequency of indels (sum of all mutant variants combined)

[0318] In summary, deep sequencing analysis showed that for two of the three designs about 50-90% of the PDS sequences have deletions of a few (1-14) nucleotide deletion close to the PAM. Reference sequences for PDS11 and PDS18, not including said deletions, are shown below in Table 4.

Table 4. Reference sequences for GmPDSll and GmPDS18

[0319] Plants with confirmed somatic edits were further grown until maturity (reproductive growth stage) in the same growth conditions. Systemic infection of BPMV was observed throughout development. When the plants reached the R8 growth stage, pods were collected, and the seeds were dried and stored at normal seed storage conditions.

Example 6: Genotyping of soybean progeny for inherited edits using amplicon sequencing

[0320] For VIGE to have wide applicability for gene editing, produced edits would need to be transmitted to progeny. To demonstrate the heritability of the method, seeds from seven PDS edited plants (from Example 4) were sown, and leaf tissue (10 days old) was sampled for target sequencing (and viral load determination).

[0321] Genomic DNA (purified with CleanXtract device using CleanNGS magnetic beads; CleanNA; The Netherlands) was used to assess PDS editing rates by genotyping of on-target gene editing outcomes after amplification of edited regions of interest with specific primers (Table 5; SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68). To better understand segregation rates of the two PDS genes independently, at this stage we have chosen to perform amplicon sequencing analysis for the two genes autonomously. Table 5. Amplicon sequencing primers used during first-round PCR amplification of the individual PDS genes.

[0322] Deep sequencing analysis of seven PDS progeny plants confirmed that seedlings from the progeny contain edits in at least one of the two PDS target sites. Sequencing data for part of this experiment are summarized in Table 6. In short, we found that each plant from progeny showing editing contains 50% edited PDS sequences in either one or both genes, and separate examination of PDS homologs shows that both seem equally efficiently edited, as expected given that both genes have identical target sequence.

[0323] Indel analysis shows that within one plant, all mutations belonged to the same cluster variant as expected from mutations inherited from one initial germline cell(s). Mostly, inherited mutations are very variable across plants raging from 1 to 26 nt deletion.

[0324] In summary, amplicon sequencing analysis showed that all seven plants analysed from PDS progeny produced at least one inherited edited seed, and that no virus was detected in the next generation of edited plants. This indicates that editing has occurred in meristematic cells.

Table 6. Overview table summarizing deep sequencing results for progeny of three plants for PDS11 and PDS18 editing rates (corresponding indel mutations are described for each of the genes.

[0325] To further confirm that the presently described techniques can be used in a method for production of inheritable edits, plants with confirmed JAG1 somatic edits were further grown until maturity (reproductive growth stage), in the same growth conditions described in the examples above. [0326] The following BPMV-RNA2 constructs were used for production of JAG1 edited plants via viral delivery.

1. pIN5744 containing the insertion of crRNA_l_GmJAGl specific for the soybean gene homologous to Arabidopsis JAGGED (JAG1; Glyma.20g 116200). crRNA_l_GmJAGl was composed of the target sequence (CCGGATGAAGAGGTATGGTCTT; SEQ ID NO: 72); flanked by DR-DR sequences.

2. pIN5743 containing the insertion of crRNA_2_GmJAGl specific for the soybean gene homologous to Arabidopsis JAGGED (JAG1 ; Glyma20gl 16200). crRNA_2_GmJAGl was composed of the target sequence (CCGGATGAAGAGGTATGGTCTT; SEQ ID NO: 72); flanked by DR-DR, all preceded by catRNA.

[0327] Systemic infection of BPMV was observed and editing of JAG1 was confirmed via amplicon sequencing. This followed the same procedure as used in Example 5, with the exception that here JAG1 AmpSeq Primers: CTCTCTCTTATGACTTTGTTG (forward; SEQ ID NO: 74) and GTGTGTGATTGTGAAATAGA (reverse; SEQ ID NO: 75) were used. Editing rates are summarized below in Table 7.

Table 7. Overview table summarizing deep sequencing results for JAG1 somatic editing. Values shown depict frequency of indels (sum of all mutant variants combined)

[0328] When the plants reached the R8 growth stage, pods were collected, and the seeds were dried and stored at normal seed storage conditions. These seeds were later germinated in petri dishes containing two layers of Whatman paper saturated with water.

[0329] Approximately 48 seeds from 16 JAG1 -edited plants (produced as described in Example 3 and Example 4) were sown, and seedling tissue (3 days old) was sampled for target sequencing. Genotyping of soybean progeny for JAG1 inherited edits was performed using amplicon sequencing. Six of the sixteen plants produced at least one inherited edited seed, resulting in 12 seeds with inherited JAG1 edit(s), including one seed that exhibited biallelic edits to JAG1. Table 8 below summarizes these results.

Table 8. Overview table summarizing heritability results for JAGl-edited plants

Example 7: Achieving viral-induced gene editing at multiple targets simultaneously (multiplex editing)

[0330] To test virus-induced gene editing (VIGE) directed to multiple targets in parallel, BPMV was engineered to carry multiple different CRISPR guide RNAs targeting sites within target soybean genes. The selected target combination described here comprises GmFT4 (Flowering Focus T4), GmFTla (Flowering Focus Tla), and GmPDS (phytoene desaturase).

[0331] For guide arrays targeting all three genes (GmFT4, GmFTla, and GmPDS) in parallel, cloning was performed in a similar manner for each guide array, at the same RNA2 location. The following constructs were produced:

1. pIN5414 containing the insertion of crRNA_l_FT4_FTla_PDS specific for the FT4, FTla, and PDS soybean genes. crRNA_l_FT4_FTla_PDS was composed of the target sequences (SEQ ID NO: 63 (GmPDSl l); SEQ ID NO: 64 (GmPDS18); SEQ ID NO: 78 (FTla), and SEQ ID NO: 79 (FT4)) flanked by DR-DR, all preceded by one single catRNA.

2. pIN5734 containing the insertion of crRNA_2_FT4_FTla_PDS specific for the FT4, FTla, and PDS soybean genes. crRNA_2_FT4_FTla_PDS was composed of the target sequences (SEQ ID NO: 63 (GmPDSl l); SEQ ID NO: 64 (GmPDS18); SEQ ID NO: 78 (FTla), and SEQ ID NO: 79 (FT4)); flanked by DR-DR. Reference sequences for PDS have been listed previously in Table 4 and reference sequences of FT la and FT4 genes from soybean are listed in Table 9.

Table 9. Reference sequences for GmFTla and GmFT4

[0332] These constructs were used to produce primary inoculum as described in Example 3, and subsequently that inoculum was used to infect Cas-overexpressing lines produced as described earlier (Example 2). The infection method used consisted of leaf-rubbing inoculation of the first leaf with infectious lysate, as already described in Example 4.

[0333] Cas-transgenic soybean plants inoculated with each of the multiplex guide array constructs were then genotyped for gene knockout of the three targets using amplicon-sequencing, together with mock and BPMV-WT control samples.

[0334] Most edited variants for any of the three targets showed deletions around the crRNA target region from one to nine nucleotides (up to 26 nt in a few cases) but no nucleotide insertion. Summarized results across replicates are shown in Table 10. Table 10. Overview table summarizing amplicon sequencing results for the multiple targets using pIN5414. Values shown depict frequency of indels (sum of all mutant variants combined)

[0335] To further demonstrate that the method described here can also be used to produce stable plants with edits at multiple targets (multiplex inheritance), progeny analysis has been conducted for the 3-target edited plants produced above.

[0336] Most progeny from the multiplex edited plants contained PDS-edits only, but two plants showed to contain edited FT la additionally to PDS (PDS11 and/or PDS18). One other plant showed to contain a FT4-only edit. All results are summarized in Table 11.

Table 11. Overview table summarizing heritability results for multiplex edited plants

Example 8: Virus-induced promoter editing (VIPE): Using virus-delivery for producing promoter edits and fine-tuning the expression of target genes

[0337] Editing plant promoters to create cis-regulatory alleles with altered gene expression levels is an innovative approach in plant biotechnology that focuses on modifying the regulatory regions of genes rather than the coding sequences. Promoters contain cis-regulatory elements (CREs) that control when, where, and how much a gene is expressed. By using tools like virus-induced promoter editing, these CRE sites can be edited to enhance or reduce the expression of target genes, enabling the fine-tuning of trait genes. This method holds significant promise for improving plant performance but also presents challenges, including ensuring the exact edits are obtained and control the potential for variable and unpredictable phenotypes, for example due to unclear length and regulation signal response elements in different promoters which could require multiplex editing, large fragment deletion, and further complex strategies.

[0338] Promoter edits were produced by virus-delivery of guide RNAs targeting the promoters of two targets: AlPlOa and AlPlOb. The following constructs were used:

[0339] pIN6437 contained crRNA_l_AIP10a specific for GmAIPlOa promoter (Glyma.07G021400) from soybean. crRNA_l_AIP10a was composed of target sequence (spacer: SEQ ID NO: 76; Table 12) flanked by two direct repeats (DR-spacer-DR; also referred to as DR-DR design or configuration). [0340] pIN6439 contained crRNA_l_AIP10b specific for GmAIPlOb promoter (Glyma.08G220400) from soybean. crRNA_l_AIP10b was composed of target sequence (spacer: SEQ ID NO: 77; Table 12) flanked by two direct repeats (DR-spacer-DR; also referred to as DR-DR design or configuration).

Table 12. Reference sequences for constructs and guides targeting AlPlOa and AlPlOb

[0341] These constructs were used to produce primary inoculum as described in Example 3, and subsequently that inoculum was used to infect Cas-overexpressing lines produced as described earlier (Example 2). The infection method used consisted of leaf-rubbing inoculation of the first leaf with infectious lysate, as already described in Example 4.

[0342] Cas-transgenic soybean plants inoculated with each of the those constructs were then genotyped for promoter editing of the corresponding promoter using amplicon-sequencing, together with mock and BPMV-WT control samples.

[0343] Most edited variants for any of the three targets showed deletions around the crRNA target region from five to 15 nucleotides but no nucleotide insertion. Summarized results across replicates are shown in Table 13.

Table 13. Overview table summarizing amplicon sequencing results for promoter edits of AlPlOa and AlPlOb. Values shown depict frequency of indels. Five most abundant indels are listed under columns “varl” - “var5”

Example 9: Use of VIGE to achieve precise editing, such as replacements and insertions

[0344] Described below is a method for precise gene editing using, for example, BPMV as described in Examples 1-8.

[0345] This method will enable precise genetic modifications, including the insertion or replacement of DNA fragments, by harnessing the natural ability of viruses to infect plant cells. The process typically involves engineering a plant virus such as Bean Pod Mottle Virus (or Tobacco Rattle Virus, or others) to carry components of the editing machinery, including a guide RNA that targets a specific genomic location and a donor DNA template that contains the desired genetic fragment for insertion or replacement.

[0346] Once the virus infects the plant cells, the nuclease enzyme (in some embodiments, a Cas nuclease), guided by the gRNA, will create a double-stranded break at the precise genomic location targeted. For fragment insertion, a donor nucleic acid (DNA or RNA) template carried by the virus will be used to insert a new gene or gene variant at the cut site via homologous recombination (HR). Similarly, for gene replacement, the donor DNA will contain a modified version of the gene, allowing the plant’s natural repair mechanisms to replace the original gene sequence with the new one.

[0347] This approach will enable precise editing at specific genomic sites, providing a tool for genetic improvement.

Example 10: Using the virus as a delivery tool for the CAS nuclease or other editing tools (along with crRNA or gRNAs)

Introduction [0348] Viral vectors developed for soybean functional genomics research offer limited cargo capacity and inserts larger than 2 kb are often lost during viral replication in the plant host (Khakhar, A. and D. Voytas. RNA Viral Vectors for Accelerating Plant Synthetic Biology. Frontiers in Plant Science. 2021. V12). To circumvent this problem, the previous examples described herein have made use of “editor lines” that constitutively express a CRISPR/Cas nuclease. Guide RNAs (gRNAs) are delivered in planta through a viral vector derived from Bean Pod Mottle Virus (BPMV) as discussed in the previous examples.

[0349] The following example is a method to use a viral vector derived from BPMV to deliver a full set of gene editing reagents including the RNA-guided nuclease (to nuclease-free plant cells). This avoids the need for a Cas-editor expressing line and allows for flexibility in the type of Cas that can be delivered, leading to greater possibilities to edit a variety of target genes. Moreover, a variety of viral vectors could potentially be used to carry different cargo, thus providing a flexible system for multiplex editing in a heritable fashion.

[0350] In this example, a Mini Cas will be employed to enable VIGE in wild-type soybean plants. While a specific Cas enzyme is used in this example, a person skilled in the art would appreciate that other nucleases may be used, and that a variety of Mini Cas’s may be used. For example, TnpB/ISDra2 may be used. Guide RNAs would be varied accordingly. Similarly, where BPMV RNA2 is used, it will be appreciated that it is also possible to use BPMV RNA1.

[0351] Casl2f belongs to Type V Cas system characterized by its compact nuclease size ranging from 422 to 603 amino acids. Casl2f was initially identified to possess activity specific to singlestranded DNA cleavage as described by Harrington et al. in 2018 (Harrington et al. Programmed DNA destruction by miniature CRISPR-Casl4 enzymes. Science, 2018. Nov 16; 362(6416):839-842), yet subsequent research extended this functionality to the cleavage of double-stranded DNA (Karvelis et al. PAM recognition by miniature CRISPR-Casl2f nucleases triggers programmable double-stranded DNA target cleavage, Nucleic Acids Research. 2020. 48(9): 5016-5023).

[0352] A novel application of SpCasl2f in agricultural biotechnology has been evidenced by successful targeted mutagenesis in maize, achieved through the application of multiple heat shock treatments at 45°C (Bigelyte, G. et al. Miniature type V-F CRISPR-Cas nucleases enable targeted DNA modification in cells. Nature Comm. 2021. 12(6191)). Furthermore, Sukegawa et al. 2023 (Genome editing in rice mediated by miniature size Cas nuclease SpCasl2f. Front. Genome Ed. Sec. Genome Editing in Plants. March. Vol. 5) demonstrated the suitability of Casl2f for targeted mutagenesis in rice calli and suggested that the enzyme be adopted for VIGE applications, such as the use of heritable VIGE in soybean.

[0353] Accordingly, conditions that are suitable for soybean will be tested.

Methods

[0354] To this end, DNA fragments of SpCasl2f, preferably soybean codon-optimized SpCasl2f, including a nuclear localization signal (NLS) at the N terminus, as well as BPMV-vector specific overhangs at 5’ and 3’ ends for Gibson cloning, will be synthesized by Twist Bioscience (San Francisco, California, USA). This fragment will be introduced into a BPMV-RNA2 viral vector by means of standard restriction ligation cloning. To enable processing of the SpCasl2f gRNA from the viral genome, said gRNA may be linked to in any manner set forth in the previous examples, but for purposes of this example, the gRNA will be linked in a 5’ to 3’ direction with tRNAs in the manner shown in FIG. 3. The SpCasl2f gRNA will be designed to target the PDS paralogs of soybean and introduced into the BPMV-SpCasl2f vector in a second round of Gibson cloning. The integrity of the complete vector (pBPMV2-SpCasl2f_tRNA-GmPDS-tRNA) will be validated by full-plasmid sequencing using short-read and/or long-read sequencing. While the gRNAs used will be designed to be specific for SpCasl2f, other gRNAs can be designed, if an alternative Mini Cas is chosen.

[0355] After upscaling and purification (e.g. a maxiprep of plasmid DNA performed with a kit, or using a commercially supplied maxiprep), both unmodified BPMV-RNA2 and BPMV pBPMV2- SpCasl2f_tRNA-GmPDS-tRNA will be applied to wild-type soybean plants using the procedures described in the examples above. A suitable growing regime will be used for replication of BPMV and gene editing by means of virally encoded SpCasl2f. Other suitable guide RNAs directed to other genes of interest will also be tested in this system: this will allow assessment of the importance of the target gene of interest itself in VIGE success and confirm the success of multiplex editing using this method. [0356] During this procedure, BPMV vectors will be first rub-inoculated to wild-type plants to produce infectious inoculum. This inoculum will be tested for the presence of the virus (via qPCR) and subsequently applied to wild-type plants where editing will be expected to occur. This two-step procedure will ensure that the virus will replicate to a high viral titre in wild- type plants, which will significantly contribute to increased levels of editing efficiency. Infected plants can be further sampled, lyophilized, and used to produce infectious lysate for later infection experiments. It is expected that this subsequent passage of replicating virus will induce more potent editing than the first rub-inoculation.

[0357] When using SpCasl2f-expressing derivatives of BPMV-RNA2, it is expected that edits will co-occur with viral infection. Therefore, plants that are symptomatic for BPMV infection and GmPDS editing (e.g. leaf mottling and bleaching), will be sampled 3 to 4 weeks after rub-inoculation. Next, viral infection will be confirmed by means of RT-qPCR performed on symptomatic plants vs mock-infected plants. The integrity of replicating SpCasl2f-expressing of BPMV-RNA2 derivatives will be further validated through sequencing. Additional qPCR assays will be performed to 1) ensure the presence of replicating virus, and 2) inspect the integrity of the SpCasl2f-expressing BPMV-RNA2 derivatives, and 3) check the expression of SpCasl2f. Plant samples that satisfy all conditions - namely, high viral titer, intact genomic architecture that retains the SpCasl2f open reading frame (ORF), and expression of SpCasl2f, will be sampled and subjected to amplicon-sequencing to examine the presence and frequency of edits, and evaluate editing efficiency. These plants will be successfully brought to maturity, and the resulting progeny will be evaluated for the presence of inheritable editing.

Example 11: Use of VIGE in combination with gene silencing for enhancing gene editing efficiency [0358] The presently described techniques can be combined in a method for increasing the efficiency of gene editing, by using virus-induced gene silencing for suppressing virus-response mechanisms, enhance DNA accessibility, prevent edit correction or others.

[0359] The method will include the following steps:

[0360] Step 1: Selection of a suitable virus, such as the BPMV (Bean pod mottle virus).

[0361] Step 2: Preparation of viral cargo: Preparation of a suitably sized gene fragment, such as a 200 bp fragment, of a gene involved in repressing viral defense. This gene could be, for example, the RDR2 gene, the RDR6 gene, the Dicer-like (DCL) gene, and/or at least one other RNA-dependent RNA Polymerase (RdRP) gene. Other examples of genes that may be silenced or suppressed to increase the efficiency of gene editing include double-stranded RNA-binding protein (DRB) genes, the DRB4 gene, genes for Dicer-like proteins, HEN1 (HUA ENHANCER 1) methyltransferase gene, the SGS3 gene, etc.

[0362] Step 3: Introduction of the viral cargo into plants: The viral cargo will be introduced into the plants using rub-inoculation or standard Agrobacterium-mediated transformation method described in previous examples. This method will involve the use of a disarmed strain of Agrobacterium tumefaciens, which will carry the viral cargo into the plant cells.

[0363] Step 4: Silencing of target genes: The introduction of that viral cargo into the plant will lead to suppression of RNA-silencing genes. This silencing can disrupt critical viral defense mechanisms and increases efficiency of gene editing. Alternatively, depending on the specific targeted gene, silencing can result in suppression of other specific cellular mechanisms which lead to increased CRISPR-mediated gene editing efficiency. These mechanisms often involve pathways related to DNA repair, cell cycle regulation, and the cellular immune response. For example, inhibiting DNA repair pathways such as Non-Homologous End Joining (NHEJ) and Mismatch Repair (MMR) can increase the precision of gene edits. Also, blocking NHEJ shifts DNA repair towards Homology-Directed Repair (HDR), a more accurate process for incorporating specific genetic changes. Similarly, reducing MMR activity prevents the correction of desired edits, thereby improving the incorporation of intended changes during HDR. Additionally, cell cycle modulation by arresting cells in the S or G2 phases, where HDR is most active, can further increase the precision of gene edits. Also, chromatin structure plays a critical role in DNA accessibility; suppressing enzymes that condense chromatin, such as histone deacetylases (HDACs), can make target DNA more accessible to nucleases, leading to more effective gene editing.

[0364] Step 5: Monitoring and evaluation: The efficiency of the gene editing process will be monitored by tracking editing rates via amplicon-sequencing and monitoring the expression levels of the target genes before and after the introduction of the viral cargo. This will be done using standard molecular biology techniques, as are known in the art. [0365] This embodiment provides a feasible method for increasing the efficiency of virus-induced gene editing in plants. It also addresses the technical problems of off-target effects, potential integration of viral sequences into the plant genome, and the low efficiency of current gene editing methods.

FIRST SET OF ENUMERATED EMBODIMENTS

Embodiment 1A. A method of editing a genomic target in a scion comprising grafting the scion onto a rootstock expressing a Cas nuclease, wherein the rootstock comprises nucleic acid encoding the Cas nuclease fused to a meristem transport segment (MTS); and delivering a guide RNA for the Cas nuclease to the scion by virus-mediated delivery.

Embodiment 2A. The method of embodiment 1A, further comprising transforming the rootstock with nucleic acid encoding the Cas nuclease prior to grafting.

Embodiment 3 A. The method of any one of embodiments 1A-2A, wherein the scion comprises a leaf, a shoot, a stem, and/or a meristem.

Embodiment 4A. A method of editing a genomic target in the meristem of a plant comprising transforming the root of the plant with nucleic acid encoding a Cas nuclease; and delivering a guide RNA for the Cas nuclease to a leaf, a shoot, a stem, and/or meristem of the plant by virus-mediated delivery, wherein the nucleic acid encoding the Cas nuclease is fused to a meristem transport segment (MTS).

Embodiment 5 A. The method of any one of embodiments 1A-4A, wherein the guide RNA is fused to a meristem transport segment (MTS).

Embodiment 6A. The method of any one of embodiments 3A-5A, wherein delivery of the guide RNA comprises inoculating the leaves, shoot, stem, and/or meristem with a composition comprising a recombinant plant virus that comprises the guide RNA or a polynucleotide encoding the guide RNA.

Embodiment 7A. The method of embodiment 6A, wherein the composition comprising the recombinant plant virus is infectious sap.

Embodiment 8A. The method of embodiment 7A, wherein the infectious sap is provided by inoculating the leaves of a host plant with an infectious cDNA plasmid and collecting infectious sap from the host plant. Embodiment 9A. The method of embodiment 8A, wherein the method further comprises selecting one or more intermediate host plants that are infected with viruses carrying intact cargo comprising the guide RNA, raising the selected plants, and collecting the infectious sap from the selected plants.

Embodiment 10A. The method of embodiment 9 A, wherein intermediate host plants that are highly infected with viruses are identified by detecting expression levels of viral coat protein, and/or detecting the presence of intact viral cargo.

Embodiment 11 A. The method of embodiment 10A, wherein intermediate host plants that are highly infected with viruses are identified by measuring levels of viral coat protein-encoding mRNA in the intermediate host plants by RT-qPCR.

Embodiment 12A. The method of any one of embodiments 10A, wherein assaying the presence of intact viral cargo comprises sequencing infectious cDNAs in the intermediate host plant to confirm that no spontaneous mutations have accumulated in the cargo to be delivered.

Embodiment 13 A. The method of embodiment 7A, wherein the infectious sap is provided by performing leaf infiltration of tobacco leaves with bacteria comprising an infectious cDNA plasmid, and collecting infectious sap from the tobacco leaves.

Embodiment 14A. The method of any one of embodiments 6A-13A, wherein delivery of the guide RNA comprises direct leaf rub inoculation with infectious sap.

Embodiment 15 A. The method of any one of embodiments 1A-5A, wherein delivery of the guide RNA comprises transforming the root of the plant with a bacterium comprising a binary vector comprising a recombinant plant virus.

Embodiment 16A. The method of embodiment 15 A, wherein the bacterium further comprises a binary vector comprising the Cas nuclease.

Embodiment 17A. The method of any one of embodiment 1A-16A, wherein the recombinant plant virus used in the virus-mediated delivery is a negative strand RNA virus.

Embodiment 18A. The method of any one of embodiments 1A-17A, wherein the recombinant plant virus used in the virus-mediated delivery has a segmented genome. Embodiment 19A. The method of any one of embodiments 1A-17A, wherein the recombinant plant virus used in the virus-mediated delivery further comprises an expression cassette comprising a reporter gene.

Embodiment 20A. The method of embodiment 19 A, wherein the reporter gene encodes a fluorescent reporter.

Embodiment 21 A. The method of any one of embodiments 3A-14A, wherein the recombinant plant virus is capable of cell-to-cell movement.

Embodiment 22A. The method of any one of embodiments 1A-21A, wherein RNA encoding the Cas nuclease and/or the guide RNA is transported by plant vascular system.

Embodiment 23 A. The method of any one of embodiments 1A-22A, wherein RNA encoding the Cas nuclease and/or the guide RNA is transported to the scion through the xylem or the phloem.

Embodiment 24A. The method of any one of embodiments 1A-23A, wherein RNA encoding the Cas nuclease and/or the guide RNA is transported to the meristem.

Embodiment 25A. The method of embodiment 24A, wherein RNA encoding the Cas nuclease is translated in the meristem.

Embodiment 26A. The method of any one of embodiments 1A-25A, wherein the genome of one or more meristematic cells is edited.

Embodiment 27A. The method of any one of embodiments 1A-26A, wherein two or more guide RNAs for the Cas nuclease are delivered to the scion.

Embodiment 28A. The method of embodiment 27A, wherein the two or more guide RNAs are encoded by a single precursor RNA.

Embodiment 29A. The method of embodiment 28A, wherein the two or more guide RNAs are each flanked by a direct repeat.

Embodiment 30A. The method of any one of embodiments 1A-3A and 5A-29A, wherein the scion and the rootstock are different plant species. Embodiment 31 A. The method of any one of embodiments 1A-3A and 5A-29A, wherein the scion and the rootstock are the same plant species.

Embodiment 32A. The method of any one of embodiments 1A-3A and 5A-31A, wherein the scion and/or rootstock is a dicot.

Embodiment 33A. The method of any one of embodiments 4A-31A, wherein the plant is a dicot.

Embodiment 34A. The method of any one of embodiments 1A-3A and 5A-31A, wherein the scion and/or rootstock is a monocot.

Embodiment 35 A. The method of any one of embodiments 4A-31A, wherein the plant is a monocot.

Embodiment 36A. The method of any one of embodiments 1A-35A, wherein the rootstock, scion, and/or plant is soy, canola, alfalfa, corn, oat, sorghum, sugarcane, banana, or wheat.

Embodiment 37A. The method of any one of embodiments 1A-36A, 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.

Embodiment 38A. The method of embodiment 37A, 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 39A. The method of embodiment 37A, wherein the TLS comprises the nucleotide sequence set forth in SEQ ID NO: 29 or 30.

Embodiment 40A. The method of any one of embodiments 1A-39A, wherein the nucleic acid encoding the MTS is located 3’ of the nucleic acid encoding the Cas nuclease.

Embodiment 41 A. The method of any one of embodiments 1A-39A, wherein the nucleic acid encoding the MTS is located 5’ of the nucleic acid encoding the Cas nuclease. Embodiment 42A. The method of any one of embodiments 1A-41A, wherein the nucleic acid encoding the Cas enzyme is linked to a promoter.

Embodiment 43A. The method of embodiment 42A, wherein the promoter is active in roots and/or phloem companion cells.

Embodiment 44A. The method of embodiment 42A, wherein the promoter is the promoter of a gene selected from the group consisting of Arabidopsis WRKY6, chickpea WRKY31, carrot MYB113, corn GLU1, strawberry RB7-type TIP-2, and banana TIP2-2, or the promoter of an orthologous gene thereof.

Embodiment 45A. The method of embodiment 42A, 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 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 JMJ 18 promoter, and a phloem protein 2 (PP2) promoter.

Embodiment 46A. The method of embodiment 42A, wherein the promoter is a constitutive promoter.

Embodiment 47A. The method of embodiment 47A, wherein the constitutive promoter is a ubiquitin promoter.

Embodiment 48A. The method of any one of embodiments 1A-47A, wherein the Cas nuclease is codon-optimized for expression in dicots.

Embodiment 49A. The method of any one of embodiments 1A-47A, wherein the Cas nuclease is codon-optimized for expression in monocots.

Embodiment 50A. The method of any one of embodiments 1A-47A, wherein the Cas nuclease is codon-optimized for expression in corn, soy or wheat.

Embodiment 51 A. The method of any one of embodiments 1A-50A, wherein the method comprises delivering two or more, three or more, four or more, or five or more guide RNAs. Embodiment 52A. The method of embodiment 51 A, wherein the two or more, three or more, four or more, or five or more guide RNAs are each joined to an MTS.

Embodiment 53A. The method of any one of embodiments 1A-52A, wherein the Cas nuclease is selected from the group consisting of Cas9, Casl2a (Cpfl), Casl2e (CasX), Casl2d (CasY), C2cl, C2c2, C2c3, Casl2h, Casl2i, and Casl2j.

Embodiment 54A. The method of any one of embodiments 1A-52A, wherein the Cas nuclease is associated with a reverse transcriptase.

Embodiment 55A. The method of embodiment 54A, wherein the Cas nuclease is fused to the reverse transcriptase.

Embodiment 56A.The method of any one of embodiments 54A-55A, wherein the guide RNA comprises at its 3’ end a priming site and an edit to be incorporated into the genomic target.

Embodiment 57A. The method of any one of embodiments 1A-56A, wherein the Cas nuclease is a Cas nickase.

Embodiment 58A. The method of embodiment 57A, wherein the Cas nickase is a Cas9 nickase or a Cas 12 nickase.

Embodiment 59A. The method of any one of embodiments 57A-58A, wherein the Cas nickase comprises mutation in one or more nuclease active sites.

Embodiment 60A. The method of any one of embodiments 1A-59A, wherein the plant further comprises nucleic acid encoding a detectable marker fused to a nucleic acid encoding an MTS.

Embodiment 61 A. The method of any one of embodiments 1A-60A, wherein the guide RNA comprises a 5 -methylcytosine group.

Embodiment 62A. The method of any one of embodiments 5A-61A, wherein the nucleic acid encoding the guide RNA and the MTS is located between two ribozyme sequences.

Embodiment 63A. The method of embodiment 62A, 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 64A. The method of any one of embodiments 5A-63A, wherein 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.

Embodiment 65A. The method of any one of embodiments 5A-64A, wherein the nucleic acid encoding the guide RNA and the MTS further comprises a terminator.

Embodiment 66A. The method of embodiment 65A, wherein the terminator is a U6 terminator.

Embodiment 67A. The method of any one of embodiments 1A-66A, further comprising retrieving a progeny of the scion or the plant, wherein the progeny has an altered genome.

Embodiment 68A. The method of any one of embodiments 1A-67A, wherein 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.

Embodiment 69A. The method of embodiment 68A, 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 70A. The method of embodiment 68A, 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 71 A. The method of any one of embodiments 1A-53A and 57A-70A, wherein the method further comprises delivering a donor template DNA to the plant by virus-mediated delivery. Embodiment 72A. The method of embodiment 71 A, wherein a sequence from the donor template DNA is incorporated into the genome of the scion.

Embodiment 73A. The method of embodiment 72A, wherein the donor template DNA is delivered to the scion using the same viral vector as the gRNA.

Embodiment 74A. The method of embodiment 72A, wherein the donor template DNA is delivered to the scion using a different viral vector than is used to deliver the gRNA.

Embodiment 75A. The method of any one of embodiments 72A-74A, wherein the sequence from the donor template DNA is incorporated into the genome of the scion at the locus targeted by the gRNA.

Embodiment 76A. The method of any one of embodiments 71A-75A, wherein the donor template DNA confers a desired trait.

Embodiment 77A. The method of any one of embodiments 71A-76A, wherein the donor template comprises an endogenous sequence.

Embodiment 78 A. The method of any one of embodiments 71A-76A, wherein the donor template comprises an exogenous sequence.

Embodiment 79A. An edited plant produced by the method of any one of embodiments 1A-78A.

SECOND SET OF ENUMERATED EMBODIMENTS

Embodiment IB. A method of producing a heritable modification in a gene of interest in a soybean plant, the method comprising: a) infecting the soybean plant with a bean pod mottle virus (BPMV) vector carrying a guide RNA (gRNA) directed to the gene of interest; b) expressing a Cas enzyme in the soybean plant, wherein sufficient time elapses for the soybean plant’ s meristem cells to be invaded by the vector and modified; and c) thereby producing the heritable modification in the gene of interest.

Embodiment 2B. The method of embodiment IB, further comprising: d) allowing the modified meristem cells to generate a seed comprising the heritable modification; and e) collecting the seed. Embodiment 3B. The method of embodiment 2B, further comprising growing the seed.

Embodiment 4B. The method of any one of embodiments 1B-3B, wherein the Cas enzyme is delivered to the soybean plant in the BPMV vector carrying the gRNA.

Embodiment 5B. The method of any one of embodiments 1B-3B, wherein the Cas enzyme is delivered to the soybean plant by a second BPMV vector comprising nucleic acid encoding the Cas enzyme.

Embodiment 6B. The method of any one of embodiments 1B-5B, the method further comprising infecting the soybean plant with a plurality of BPMV vectors, each BPMV vector comprising a gRNA and/or the Cas enzyme.

Embodiment 7B. The method of embodiment 6B, wherein the plurality of BPMV vectors co-infect the soybean plant simultaneously or infect the soybean plant in more than one round of infection.

Embodiment 8B. The method of any one of embodiments 1B-7B, wherein the soybean plant overexpresses the Cas enzyme.

Embodiment 9B. The method of any one of embodiments 1B-8B, wherein the BPMV vector comprises BPMV RNA2.

Embodiment 10B. The method of embodiment 9B, wherein the gRNA is linked 3’ to the BPMV RNA2.

Embodiment 11B. The method of any one of embodiments 1B-10B, wherein the BPMV vector carries at least two gRNAs, at least three gRNAs, at least four gRNAs, at least five gRNAs, at least six gRNAs, at least seven gRNAs, or at least eight gRNAs.

Embodiment 12B. The method of embodiment 11B, wherein each gRNA is directed to a different gene of interest in the soybean plant.

Embodiment 13B. The method of embodiment 1 IB, wherein each gRNA is directed to the same gene of interest. Embodiment 14B. The method of any one of embodiments 1B-13B, wherein the Cas enzyme is a nuclease is selected from the group consisting of Cas9, Casl2f, Casl2a (Cpfl), Casl2e (CasX), Cas 12d (CasY), C2cl, C2c2, C2c3, Casl2h, Casl2i, and Casl2j.

Embodiment 15B. The method of any one of embodiments 1B-14B, wherein the Cas enzyme is a Cas nickase.

Embodiment 16B. The method of embodiment 15B, wherein the Cas nickase is a Cas9 nickase or a Cas 12 nickase.

Embodiment 17B. The method of any one of embodiments 15B-16B, wherein the Cas nickase comprises mutation in one or more nuclease active sites.

Embodiment 18B. The method of any one of embodiments 1B-17B, wherein the Cas enzyme is heterologous to the soybean plant.

Embodiment 19B. The method of any one of embodiments 1B-18B, wherein the gRNA and the Cas enzyme form a complex and introduce a single- or double-stranded break in the sequence of the gene of interest.

Embodiment 20B. The method of any one of embodiments 1B-19B, wherein the BPMV vector comprises from 5’ to 3’: i. a first ribozyme sequence; ii. a direct repeat; iii. a spacer sequence complementary to the gene of interest; and iv. a second ribozyme sequence, wherein the first ribozyme sequence and the second ribozyme sequence are the same or different sequences.

Embodiment 21B. The method of any one of embodiments 1B-19B, wherein the BPMV vector comprises from 5’ to 3’: i. a first direct repeat; ii. a spacer sequence complementary to the gene of interest; and iii. a second direct repeat.

Embodiment 22B. The method of any one of embodiments 1B-19B, wherein the BPMV vector comprises from 5’ to 3’: i. a RNase-resistant caged truncated pre-tRNA-like crRNA (catRNA); ii. a first direct repeat; iii. a spacer sequence complementary to the gene of interest; and iv. a second direct repeat.

Embodiment 23B. The method of any one of embodiments 20B-22B, wherein the BPMV vector further comprises the Cas enzyme.

Embodiment 24B. The method of any one of embodiments 1B-23B, wherein the gRNA is processed by a ribozyme selected from the group consisting of a hammerhead ribozyme, a hepatitis delta virus (HDV) ribozyme, a Csy4, and a tRNA processing enzyme.

Embodiment 25B. The method of any one of embodiments 1B-24B, the method further comprising screening the soybean plant for success in infecting the soybean plant, said screening comprising a visual assessment of the soybean plant for desired phenotype.

Embodiment 26B. The method of any one of embodiments 1B-25B, wherein the gRNA is directed to a regulatory or coding sequence contributing to a trait selected from the group consisting of: photosynthetic ability or efficiency; yield or fertility; seed number; disease or pest resistance; herbicide or pesticide tolerance; abiotic stressor tolerance; fruit morphology; fruit nutrition; fruit ripening; number of seeds per pod; and leaf size.

Embodiment 27B. The method of any one of embodiments 1B-26B, wherein infecting the soybean plant comprises applying an inoculum comprising the BPMV vector carrying the gRNA.

Embodiment 28B. The method of any one of embodiments 1B-27B, wherein infecting the soybean plant comprises inoculating the soybean plant’s leaves, shoot, stem, roots, or other vegetative tissue with a composition comprising the BPMV vector carrying the gRNA.

Embodiment 29B. The method of embodiment 27B, wherein the inoculum is infectious sap.

Embodiment 30B. The method of embodiment 29B, wherein the infectious sap is provided by inoculating a first host plant with an infectious cDNA plasmid and collecting infectious sap from the first host plant, the cDNA plasmid comprising: i. the BPMV vector; ii. the gRNA; and/or iii. the Cas enzyme. Embodiment 31B. The method of embodiment 30B, wherein the method further comprises: a) selecting the first host plant that is highly infected with intact viral cargo comprising the BPMV vector, the gRNA, and/or the Cas enzyme; b) raising the selected plant; and c) collecting the infectious sap from the selected plant.

Embodiment 32B. The method of embodiment 30B or 3 IB, the method further comprising inoculating a second host plant with the infectious sap from the first host plant.

Embodiment 33B. The method of embodiment 31B, wherein the first host plant that is highly infected is identified by detecting expression levels of viral coat protein, and/or detecting the presence of intact viral cargo.

Embodiment 34B. The method of embodiment 3 IB, wherein the first host plant that is highly infected is identified by measuring levels of viral coat protein-encoding mRNA in the first host plant by RT- qPCR.

Embodiment 35B. The method of embodiment 33B, wherein detecting the presence of intact viral cargo comprises sequencing infectious cDNA in the first host plant.

Embodiment 36B. The method of embodiment 29B, wherein the infectious sap is provided by performing leaf infiltration of soybean or non-soybean leaves with bacteria comprising an infectious cDNA plasmid, and collecting infectious sap from the soybean or non-soybean leaves.

Embodiment 37B. The method of any one of embodiments 1B-36B, wherein infecting the soybean plant comprises direct leaf rub inoculation with infectious sap.

Embodiment 38B. A method for producing a meristem cell having a targeted genomic modification, the method comprising: a) delivering a BPMV viral vector carrying a gRNA to a meristem cell, wherein the meristem cell expresses a Cas enzyme; b) allowing the gRNA and the Cas enzyme to modify the meristem cell; and c) thereby producing the meristem cell having the targeted genomic modification.

Embodiment 39B. A method for producing soybean seed comprising a targeted genomic modification, the method comprising: a) delivering a BPMV viral vector carrying a gRNA to a soybean meristem cell, wherein the soybean meristem cell expresses a Cas nuclease; wherein the gRNA and the Cas nuclease modifies the soybean meristem cell; wherein the soybean meristem cell produces a soybean germline that forms seed; and b) thereby producing soybean seed having the targeted genomic modification.

Embodiment 40B. The method of any one of embodiments 1B-39B, wherein the method further comprises delivering a donor template DNA to the soybean plant.

Embodiment 41B. The method of embodiment 40B, wherein the donor template DNA is delivered by infecting the soybean plant with a BPMV vector.

Embodiment 42B. The method of embodiment 40B or 41B, wherein a sequence from the donor template DNA is incorporated into the genome of the soybean plant.

Embodiment 43B. The method of embodiment 42B, wherein the sequence from the donor template DNA is incorporated into the genome of the soybean plant at the gene of interest.

Embodiment 44B. The method of embodiment 41B, wherein the donor template is linked to the gRNA.

Embodiment 45B. The method of embodiment 41B, wherein the donor template DNA is delivered to the soybean plant using a different BPMV vector than the BPMV vector carrying the gRNA.

Embodiment 46B. The method of any one of embodiments 40B-45B, wherein the donor template DNA confers a desired trait.

Embodiment 47B. The method of any one of embodiments 40B-46B, wherein the donor template comprises an endogenous sequence.

Embodiment 48B. The method of any one of embodiments 40B-46B, wherein the donor template comprises an exogenous sequence.

Embodiment 49B. A bean pod mottle virus (BPMV) viral vector system comprising: i. a BPMV genome component; ii. one or more gRNA inserted into the viral vector; and iii. a direct repeat and/or self-cleaving ribozyme sequence flanking the one or more gRNA. Embodiment 50B. The viral vector system of embodiment 49B, wherein a nucleic acid modifying enzyme is overexpressed in cells that receive the viral vector system.

Embodiment 5 IB. The viral vector system of embodiment 49B, the viral vector system further comprising a nucleic acid encoding a nucleic acid modifying enzyme.

Embodiment 52B. The viral vector system of embodiment 50B or 5 IB, wherein the nucleic acid modifying enzyme is a CRISPR/Cas nuclease.

Embodiment 53B. A seed comprising an inherited modification in a gene of interest in a soybean plant, the seed produced by the method of any one of embodiments 1B-48B.

Embodiment 54B. A meristem cell having the modification produced by the method of any one of embodiments 1B-48B.

Embodiment 55B. A method of using a bean pod mottle virus (BPMV) to generate a heritable modification in a gene of interest in a soybean plant, the method comprising: a) infecting the soybean plant with a recombinant bean pod mottle virus (BPMV) carrying a guide RNA (gRNA) directed to the gene of interest; b) expressing a Cas enzyme in the plant; c) allowing sufficient time to elapse for the virus to invade the soybean plant’s meristem cells; and d) thereby using BPMV to generate the heritable modification.

Embodiment 56B. A method for making a heritable genomic modification at a target site in a soybean plant, the method comprising: a) delivering a BPMV vector comprising the sequence of BPMV RNA2 linked to a nucleic acid sequence encoding a guide RNA (gRNA) to a cell of the soybean plant, wherein the BPMV invades a meristem of the plant expressing a heterologous Cas nuclease and the gRNA is expressed in the same meristem; and b) allowing the gRNA and the Cas nuclease to form a complex and introduce a single- or double-strand break at the target site in the genome of the meristem’s cell or cells, thereby making a heritable genomic modification.

Embodiment 57B. The method of embodiment 55B, wherein the BPMV vector comprises from 5’ to 3’ : i. a first ribozyme sequence; ii. a direct repeat; iii. a spacer sequence directed to the target site; and iv. a second ribozyme sequence, wherein the first ribozyme sequence and the second ribozyme sequence are the same or different sequences.

Embodiment 58B. The method of embodiment 55B, wherein the BPMV vector comprises from 5’ to 3’: i. a first direct repeat; ii. a spacer sequence directed to the target site; and iii. a second direct repeat.

Embodiment 59B. The method of embodiment 55B, wherein the BPMV vector comprises from 5’ to 3’: i. a RNase-resistant caged truncated pre-tRNA-like crRNA (catRNA); ii. a first direct repeat; iii. a spacer sequence directed to the target site; and iv. a second direct repeat.

Embodiment 60B. The method of any one of embodiments 56B-59B, wherein the gRNA is processed by a ribozyme selected from the group consisting of a hammerhead ribozyme, a hepatitis delta virus (HDV) ribozyme, a Csy4, and a tRNA processing enzyme.

Embodiment 61B. The method of any one of embodiments 55B-60B, the method further comprising screening the soybean plant for successful genome modification, said screening comprising: a) visually assessing the soybean plant for desired phenotype; and/or b) sequencing of cells produced by the meristem after delivery of the BPMV vector.

THIRD SET OF ENUMERATED EMBODIMENTS

Embodiment 1C. A method of producing a heritable modification in a gene of interest in a soybean plant, the method comprising: f) infecting the soybean plant with a bean pod mottle virus (BPMV) vector carrying a guide RNA (gRNA) directed to the gene of interest; g) expressing a Cas enzyme in the soybean plant, wherein sufficient time elapses for the soybean plant’ s meristem cells to be edited; and h) thereby producing the heritable modification in the gene of interest. Embodiment 2C. The method of embodiment 1C, further comprising: i) allowing the edited meristem cells to generate a seed comprising the heritable modification; and j) collecting the seed.

Embodiment 3C. The method of embodiment 2C, further comprising growing the seed.

Embodiment 4C. The method of any one of embodiments 1C-3C, wherein the Cas enzyme is delivered to the soybean plant in the BPMV vector carrying the gRNA.

Embodiment 5C. The method of any one of embodiments 1C-3C, wherein the Cas enzyme is delivered to the soybean plant by a second BPMV vector comprising nucleic acid encoding the Cas enzyme.

Embodiment 6C. The method of any one of embodiments 1C-5C, the method further comprising infecting the soybean plant with a plurality of BPMV vectors, each BPMV vector comprising a gRNA and/or the Cas enzyme.

Embodiment 7C. The method of embodiment 6C, wherein the plurality of BPMV vectors co-infect the soybean plant simultaneously or infect the soybean plant in more than one round of infection.

Embodiment 8C. The method of any one of embodiments 1C-7C, wherein the soybean plant overexpresses the Cas enzyme.

Embodiment 9C. The method of any one of embodiments 1C-8C, wherein the BPMV vector comprises a BPMV genomic segment, optionally wherein the BPMV genomic segment is BPMV- RNA1 or BPMV-RNA2. .

Embodiment IOC. The method of embodiment 9C, wherein the gRNA is linked 5’ and/or 3’ to the BPMV-RNA2.

Embodiment 11C. The method of any one of embodiments 1C-10C, wherein the BPMV vector carries at least two gRNAs, at least three gRNAs, at least four gRNAs, at least five gRNAs, at least six gRNAs, at least seven gRNAs, or at least eight gRNAs. Embodiment 12C. The method of embodiment 11C, wherein each gRNA is directed to a different gene of interest in the soybean plant.

Embodiment 13C. The method of embodiment 11C, wherein each gRNA is directed to the same gene of interest.

Embodiment 14C. The method of any one of embodiments 1C-13C, wherein the Cas enzyme is a nuclease is selected from the group consisting of Cas9, Casl2f, Casl2a (Cpfl), Casl2e (CasX), Cas 12d (CasY), C2cl, C2c2, C2c3, Casl2h, Casl2i, and Casl2j.

Embodiment 15C. The method of any one of embodiments 1C-14C, wherein the Cas enzyme is a Cas nickase.

Embodiment 16C. The method of embodiment 15C, wherein the Cas nickase is a Cas9 nickase or a Cas 12 nickase.

Embodiment 17C. The method of any one of embodiments 15C-16C, wherein the Cas nickase comprises mutation in one or more nuclease active sites.

Embodiment 18C. The method of any one of embodiments 1C-17C, wherein the Cas enzyme is heterologous to the soybean plant.

Embodiment 19C. The method of any one of embodiments 1C-18C, wherein the gRNA and the Cas enzyme form a complex and introduce a single- or double-stranded break in the sequence of the gene of interest.

Embodiment 20C. The method of any one of embodiments 1C-19C, wherein the BPMV vector comprises: v. a first ribozyme sequence; vi. a direct repeat; vii. a spacer sequence complementary to the gene of interest; and viii.a second ribozyme sequence, wherein the first ribozyme sequence and the second ribozyme sequence are the same or different sequences.

Embodiment 21C. The method of any one of embodiments 1C-19C, wherein the BPMV vector comprises: iv. a first direct repeat; v. a spacer sequence complementary to the gene of interest; and vi. a second direct repeat.

Embodiment 22C. The method of any one of embodiments 1C-19C, wherein the BPMV vector comprises: i. a RNase-resistant caged truncated pre-tRNA-like crRNA (catRNA); ii. a first direct repeat; iii. a spacer sequence complementary to the gene of interest; and iv. a second direct repeat.

Embodiment 23C. The method of any one of embodiments 20C-22C, wherein the BPMV vector further comprises the Cas enzyme.

Embodiment 24C. The method of any one of embodiments 1C-23C, wherein the gRNA is processed by a ribozyme selected from the group consisting of a hammerhead ribozyme, a hepatitis delta virus (HDV) ribozyme, a Csy4, and a tRNA processing enzyme.

Embodiment 25C. The method of any one of embodiments 1C-24C, the method further comprising screening the soybean plant for success in infecting the soybean plant, said screening comprising a visual assessment of the soybean plant for desired phenotype.

Embodiment 26C. The method of any one of embodiments 1C-25C, wherein the gRNA is directed to a regulatory or coding sequence contributing to a trait selected from the group consisting of: photosynthetic ability or efficiency; yield or fertility; seed number; disease or pest resistance; herbicide or pesticide tolerance; abiotic stressor tolerance; fruit morphology; fruit nutrition; fruit ripening; number of seeds per pod; and leaf size.

Embodiment 27C. The method of any one of embodiments 1C-26C, wherein infecting the soybean plant comprises applying an inoculum comprising the BPMV vector carrying the gRNA.

Embodiment 28C. The method of any one of embodiments 1C-27C, wherein infecting the soybean plant comprises inoculating the soybean plant’s leaves, shoot, stem, roots, or other vegetative tissue with a composition comprising the BPMV vector carrying the gRNA.

Embodiment 29C. The method of embodiment 27C, wherein the inoculum is infectious lysate. Embodiment 30C. The method of embodiment 29C, wherein the infectious lysate is provided by inoculating a first host plant with an infectious cDNA plasmid and collecting infectious lysate from the first host plant, the cDNA plasmid comprising: i. the BPMV vector; ii. the gRNA; and/or iii. the Cas enzyme.

Embodiment 31C. The method of embodiment 30C, wherein the method further comprises: a) selecting the first host plant that is highly infected with intact viral cargo comprising the BPMV vector, the gRNA, and/or the Cas enzyme; b) raising the selected plant; and c) collecting the infectious lysate from the selected plant.

Embodiment 32C. The method of embodiment 30C or 31C, the method further comprising inoculating a second host plant with the infectious lysate from the first host plant.

Embodiment 33C. The method of embodiment 31C, wherein the first host plant that is highly infected is identified by detecting expression levels of viral coat protein, and/or detecting the presence of intact viral cargo.

Embodiment 34C. The method of embodiment 31C, wherein the first host plant that is highly infected is identified by measuring levels of viral coat protein-encoding mRNA in the first host plant by RT- qPCR.

Embodiment 35C. The method of embodiment 33C, wherein detecting the presence of intact viral cargo comprises sequencing infectious cDNA in the first host plant.

Embodiment 36C. The method of embodiment 29C, wherein the infectious lysate is provided by performing leaf infiltration of soybean or non-soybean leaves with bacteria comprising an infectious cDNA plasmid, and collecting infectious lysate from the soybean or non-soybean leaves.

Embodiment 37C. The method of any one of embodiments 1C-36C, wherein infecting the soybean plant comprises direct leaf rub inoculation with infectious lysate.

Embodiment 38C. A method for producing a meristem cell having a targeted genomic modification, the method comprising: a) delivering a BPMV viral vector carrying a gRNA to a meristem cell, wherein the meristem cell expresses a Cas enzyme; b) allowing the gRNA and the Cas enzyme to modify the meristem cell; and c) thereby producing the meristem cell having the targeted genomic modification.

Embodiment 39C. A method for producing soybean seed comprising a targeted genomic modification, the method comprising: c) delivering a BPMV viral vector carrying a gRNA to a soybean meristem cell, wherein the soybean meristem cell expresses a Cas nuclease; wherein the gRNA and the Cas nuclease modifies the soybean meristem cell; wherein the soybean meristem cell produces a soybean germline that forms seed; and d) thereby producing soybean seed having the targeted genomic modification.

Embodiment 40C. The method of any one of embodiments 1C-39C, wherein the method further comprises delivering a donor template DNA to the soybean plant.

Embodiment 41C. The method of embodiment 40C, wherein the donor template DNA is delivered by infecting the soybean plant with a BPMV vector.

Embodiment 42C. The method of embodiment 40C or 41C, wherein a sequence from the donor template DNA is incorporated into the genome of the soybean plant.

Embodiment 43C. The method of embodiment 42C, wherein the sequence from the donor template DNA is incorporated into the genome of the soybean plant at the gene of interest.

Embodiment 44C. The method of embodiment 41C, wherein the donor template is linked to the gRNA.

Embodiment 45C. The method of embodiment 41C, wherein the donor template DNA is delivered to the soybean plant using a different BPMV vector than the BPMV vector carrying the gRNA.

Embodiment 46C. The method of any one of embodiments 40C-45C, wherein the donor template DNA confers a desired trait.

Embodiment 47C. The method of any one of embodiments 40C-46C, wherein the donor template comprises an endogenous sequence. Embodiment 48C. The method of any one of embodiments 40C-46C, wherein the donor template comprises an exogenous sequence.

Embodiment 49C. A bean pod mottle virus (BPMV) viral vector system comprising: iv. a BPMV genome component; v. one or more gRNA inserted into the viral vector; and vi. a direct repeat and/or self-cleaving ribozyme sequence flanking the one or more gRNA.

Embodiment 50C. The viral vector system of embodiment 49C, wherein a nucleic acid modifying enzyme is overexpressed in cells that receive the viral vector system.

Embodiment 51C. The viral vector system of embodiment 49C, the viral vector system further comprising a nucleic acid encoding a nucleic acid modifying enzyme.

Embodiment 52C. The viral vector system of embodiment 50C or 51C, wherein the nucleic acid modifying enzyme is a CRISPR/Cas nuclease.

Embodiment 53C. A seed comprising an inherited modification in a gene of interest in a soybean plant, the seed produced by the method of any one of embodiments 1C-48C.

Embodiment 54C. A meristem cell having the modification produced by the method of any one of embodiments 1C-48C.

Embodiment 55C. A method of using a bean pod mottle virus (BPMV) to generate a heritable modification in a gene of interest in a soybean plant, the method comprising: e) infecting the soybean plant with a recombinant bean pod mottle virus (BPMV) carrying a guide RNA (gRNA) directed to the gene of interest; f) expressing a Cas enzyme in the plant; g) allowing sufficient time to elapse for the virus to invade the soybean plant’s meristem cells; and h) thereby using BPMV to generate the heritable modification.

Embodiment 56C. A method for making a heritable genomic modification at a gene of interest in a soybean plant, the method comprising: c) delivering a BPMV vector comprising the sequence of BPMV-RNA2 linked to a nucleic acid sequence encoding a guide RNA (gRNA) to a cell of the soybean plant, wherein the BPMV infects a meristem cell of the plant expressing a heterologous Cas nuclease and the gRNA is expressed in the same meristem cell; and d) allowing the gRNA and the Cas nuclease to form a complex and introduce a single- or double-strand break at the gene of interest in the genome of the meristem cell, thereby making a heritable genomic modification.

Embodiment 57C. The method of embodiment 44C, wherein the BPMV vector comprises from 5’ to 3’: v. a first ribozyme sequence; vi. a direct repeat; vii. a spacer sequence directed to the gene of interest; and viii. a second ribozyme sequence, wherein the first ribozyme sequence and the second ribozyme sequence are the same or different sequences.

Embodiment 58C. The method of embodiment 44C, wherein the BPMV vector comprises from 5’ to 3’: i. a first direct repeat; ii. a spacer sequence directed to the gene of interest; and iii. a second direct repeat.

Embodiment 59C. The method of embodiment 44C, wherein the BPMV vector comprises from 5’ to 3’: ii. a RNase-resistant caged truncated pre-tRNA-like crRNA (catRNA); ii. a first direct repeat; iii. a spacer sequence directed to the gene of interest; and iv. a second direct repeat.

Embodiment 60C. The method of any one of embodiments 56C-59C, wherein the gRNA is processed by a ribozyme selected from the group consisting of a hammerhead ribozyme, a hepatitis delta virus (HDV) ribozyme, a Csy4, and a tRNA processing enzyme.

Embodiment 61C. The method of any one of embodiments 55C-60C, the method further comprising screening the soybean plant for successful genome modification, said screening comprising: c) visually assessing the soybean plant for desired phenotype; and/or d) sequencing of cells produced by the meristem after delivery of the BPMV vector.

Claims

CLAIMS What is claimed is:

1. A method of editing a genomic target in a meristem cell of a soybean plant comprising: a) delivering a guide RNA (gRNA) directed to the genomic target to the meristem cell in the soybean plant by virus-mediated delivery; and b) delivering a Cas nuclease to the meristem cell, wherein the Cas nuclease and the guide RNA edit the genomic target in the meristem cell of the soybean plant, thereby editing the genomic target in the meristem cell.

2. A method of producing a soybean seed comprising an edited genomic target, the method comprising: a) delivering a guide RNA (gRNA) directed to the genomic target in a meristem cell of a parent soybean plant by virus-mediated delivery; and b) delivering a Cas nuclease to the meristem cell of the parent soybean plant, wherein the Cas nuclease and the guide RNA edit the genomic target in the meristem cell of the parent soybean plant, and wherein the meristem cell produces a soybean germline cell that contributes to the soybean seed, and thereby producing the soybean seed comprising the edited genomic target.

3. The method of claim 1, wherein the edited genomic target is inherited by at least one progeny or seed of the soybean plant.

4. The method of claim 1, further comprising: c) allowing the meristem cell to generate a seed comprising the edited genomic target; and d) collecting the seed.

5. The method of any one of claims 1-4, wherein virus-mediated delivery comprises using a viral vector comprising the gRNA.

6. The method of any one of claims 1-5, wherein the Cas nuclease is fused to a meristem transport segment (MTS).

7. The method of any one of claims 5-6, wherein the virus-mediated delivery comprises infecting the soybean plant with an inoculum comprising the viral vector.

8. The method of any one of claims 5-7, wherein the viral vector comprises a recombinant plant virus that comprises the guide RNA or a polynucleotide encoding the guide RNA.

9. The method of claim 8, wherein the recombinant plant virus used in the virus-mediated delivery is a virus with a segmented genome.

10. The method of any one of claims 8-9, wherein the recombinant plant virus further comprises an expression cassette comprising an endogenous visible marker gene or a reporter gene, optionally wherein the reporter gene encodes a fluorescent reporter.

11. The method of any one of claims 8-10, wherein the recombinant plant virus is capable of cell-to- cell movement.

12. The method of any one of claims 5-11, wherein the viral vector comprises bean pod mottle virus (BPMV), optionally wherein the BPMV vector comprises BPMV-RNA2 and/or BPMV-RNA1.

13. The method of claim 12, wherein the BPMV-RNA2 and/or BPMV-RNA1 is linked to, or otherwise carries, the gRNA.

14. The method of claim 12 or 13, wherein the viral vector is delivered to a leaf, shoot, stem, root, or other vegetative tissue.

15. The method of any one of claims 5-14, wherein the viral vector comprises at least two gRNAs, at least three gRNAs, at least four gRNAs, at least five gRNAs, at least six gRNAs, at least seven gRNAs, or at least eight gRNAs, optionally wherein each gRNA is directed to:

(i) a different genomic target in the soybean plant; or

(ii) a same genomic target in the soybean plant.

16. The method of any one of claims 1-15, wherein the gRNA is directed to a regulatory or coding sequence, optionally wherein the regulatory or coding sequence contributes to a trait selected from the group consisting of: photosynthetic ability or efficiency; yield or fertility; seed number; disease or pest resistance; herbicide or pesticide tolerance; abiotic stressor tolerance; fruit morphology; fruit nutrition; fruit ripening; number of seeds per pod; and leaf size.

17. The method of any one of claims 5-16, wherein infecting the soybean plant comprises inoculating the soybean plant’s leaves, shoot, stem, roots, or other vegetative tissue with a composition comprising the viral vector that contains the gRNA.

18. The method of any one of claims 1-17, wherein the Cas nuclease is delivered by virus-mediated delivery.

19. The method of any one of claims 5-18, wherein the viral vector comprises the Cas nuclease.

20. The method of any one of claims 1-19, wherein RNA encoding the gRNA and/or the Cas nuclease is delivered to the meristem cell of the soybean plant by transport from another plant tissue.

21. The method of claim 20, wherein RNA encoding the Cas nuclease is translated in the meristem cell.

22. The method of any one of claims 5-21, wherein the viral vector comprising the gRNA further comprises an RNA-guided nuclease.

23. The method of any one of claims 5-22, wherein the viral vector comprising the gRNA further comprises the Cas nuclease.

24. The method of any one of claims 5-21, wherein the Cas nuclease is delivered to the meristem cell of the soybean plant in a second viral vector comprising the Cas nuclease.

25. The method of any one of claims 5-24, the method further comprising infecting the soybean plant with a plurality of viral vectors, wherein each viral vector comprises one or more gRNA and/or the Cas nuclease.

26. The method of claim 25, wherein the plurality of viral vectors co-infect the soybean plant simultaneously or infect the soybean plant in more than one round of infection.

27. The method of any one of claims 1-26, wherein the soybean plant overexpresses the Cas nuclease.

28. The method of any one of claims 1-27, wherein the genomic target is in a scion.

29. The method of claim 28, wherein the Cas nuclease is delivered to the scion by transport from a grafted rootstock.

30. The method of claim 29, further comprising transforming the rootstock with a nucleic acid encoding the Cas nuclease prior to grafting.

31. The method of claim 29 or 30, wherein the scion and the rootstock are the same plant species or different plant species, optionally wherein the rootstock is canola, alfalfa, corn, oat, sorghum, sugarcane banana, or wheat.

32. The method of any one of claims 1-31, wherein the Cas nuclease is delivered by transport from another part of the plant through the plant vascular system.

33. The method of any one of claims 7-32, wherein the inoculum is infectious lysate, optionally wherein the infectious lysate is provided by

(a) performing leaf infiltration of soybean or non-soybean leaves with bacteria comprising an infectious cDNA plasmid, and

(b) collecting infectious lysate from the soybean or non-soybean leaves, thereby providing the infectious lysate.

34. The method of claim 33, wherein the infectious lysate is provided by inoculating a set of first host plants with at least one infectious cDNA plasmid and collecting infectious lysate from the first host plant, the cDNA plasmid comprising: i. the recombinant plant virus; ii. the gRNA; and/or iii. the Cas nuclease.

35. The method of claim 34, wherein the method further comprises: a) selecting from the set of first host plants a selected plant that is highly infected with intact viral cargo comprising the recombinant plant virus, the gRNA, and/or the Cas nuclease; b) raising the selected plant; and c) collecting the infectious lysate from the selected plant, the method optionally comprising inoculating a second host plant or a set of second host plants with the infectious lysate from the first selected plant.

36. The method of claim 35, wherein the selected plant is identified by

(a) detecting expression levels of viral coat protein, and/or detecting the presence of intact viral cargo, wherein detecting the presence of intact viral cargo optionally comprises sequencing infectious cDNA in the first host plant; and/or

(b) measuring levels of viral coat protein-encoding mRNA in the first host plant by RT-qPCR.

37. The method of any one of claims 35-36, wherein the first host plant or selected plant is Nicotiana or soybean.

38. The method of any one of claims 1-37, wherein the virus-mediated delivery comprises direct leaf rub inoculation with infectious lysate comprising the gRNA.

39. The method of any one of claims 1-38, wherein the gRNA comprises a 5 -methylcytosine group.

40. The method of any one of claims 5-39, wherein the viral vector comprises: i. a first ribozyme sequence; ii. a direct repeat; iii. a spacer sequence complementary to the gene of interest; and iv. a second ribozyme sequence, wherein the first ribozyme sequence and the second ribozyme sequence are the same or different sequences.

41. The method of any one of claims 5-39, wherein the viral vector comprises: i. a first direct repeat; ii. a spacer sequence complementary to the gene of interest; and iii. a second direct repeat.

42. The method of any one of claims 5-39, wherein the viral vector comprises: i. a RNase-resistant caged truncated pre-tRNA-like crRNA (catRNA); ii. a first direct repeat; iii. a spacer sequence complementary to the gene of interest; and iv. a second direct repeat.

43. The method of any one of claims 1-42, wherein the gRNA is processed by a ribozyme selected from the group consisting of a hammerhead ribozyme, a hepatitis delta virus (HDV) ribozyme, a Csy4, and a tRNA-derived sequence.

44. The method of any one of claims 1-43, the method further comprising screening the soybean plant for viral infection, said screening comprising a visual assessment of the soybean plant for a desired phenotype.

45. The method of any one of claims 6-44, wherein the soybean plant further comprises a nucleic acid encoding a detectable marker fused to a nucleic acid encoding the MTS, optionally wherein the nucleic acid encoding the MTS is located 3’ or 5’ of a nucleic acid encoding the Cas nuclease.

46. The method of any one of claims 6-45, 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, optionally 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.

47. The method of claim 46, wherein the TLS comprises the nucleotide sequence set forth in SEQ ID NO: 29 or 30.

48. The method of any one of claims 1-47, wherein the nucleic acid encoding the Cas nuclease is linked to a promoter, optionally wherein the promoter is active in roots and/or phloem companion cells.

49. The method of claim 48, wherein the promoter is a constitutive promoter, optionally wherein the constitutive promoter is a ubiquitin promoter.

50. The method of claim 48, wherein the promoter is selected from the group consisting of a promoter from a Arabidopsis WRKY6 gene, a promoter from a chickpea WRKY31 gene, a promoter from a carrot MYB113 gene, a promoter from a corn GLU1 gene, a promoter from a strawberry RB7-type TIP-2 gene, a promoter from a banana TIP2-2 gene, 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 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 JMJ 18 promoter, and a phloem protein 2 (PP2) promoter, or the promoter of an orthologous gene thereof.

51. The method of any one of claims 1-50, wherein the Cas nuclease is codon-optimized for expression in dicots.

52. The method of any one of claims 1-51, wherein the Cas nuclease is codon-optimized for expression in soybean.

53. The method of any one of claims 1-52, wherein the Cas nuclease is a nuclease is selected from the group consisting of Cas9, Casl2a (Cpfl), Casl2e (CasX), Casl2d (CasY), C2cl, C2c2, C2c3, Casl2h, Casl2i, and a Mini Cas.

54. The method of any one of claims 1-53, wherein the Cas nuclease is a Cas nickase, optionally wherein the Cas nickase is a Cas9 nickase or a Casl2 nickase.

55. The method of claim 54, wherein the Cas nickase comprises mutation in one or more nuclease active sites.

56. The method of any one of claims 1-55, wherein the gRNA is heterologous to the soybean plant.

57. The method of any one of claims 1-56, wherein the gRNA and the Cas nuclease form a complex and introduce a single- or double-stranded break in the sequence of the genomic target.

58. The method of any one of claims 1-57, wherein the method further comprises delivering a donor template DNA to the soybean plant, optionally wherein the donor template DNA is delivered by infecting the soybean plant with a viral vector that infects the meristem cell.

59. The method of claim 58, wherein a sequence from the donor template DNA is incorporated into the genome of the soybean plant.

60. The method of claim 59, wherein the sequence from the donor template DNA is incorporated into the genome of the soybean plant at the genomic target.

61. The method of claim 59 or 60, wherein the donor template DNA is delivered to the soybean plant using the same viral vector as the gRNA.

62. The method of any one of claims 58-61, wherein the Cas nuclease is fused to a reverse transcriptase.

63. The method of any one of claims 58-62, wherein the guide RNA comprises at its 3’ end a priming site and an edit to be incorporated into the genomic target.

64. The method of any one of claims 58-63, wherein the gRNA is a prime editing guide RNA (pegRNA).

65. The method of any one of claims 58-60 or 62-64, wherein the donor template DNA is delivered to the soybean plant using a different viral vector than the viral vector comprising the gRNA.

66. The method of any one of claims 58-65, wherein the donor template DNA confers a desired trait on the plant, and optionally wherein the donor template comprises an exogenous or endogenous sequence.

67. The method of any one of claims 1-66, wherein the virus-mediated delivery comprises transforming the root of the plant with a bacterium comprising a binary vector comprising a recombinant plant virus comprising the guide RNA or a nucleic acid encoding the guide RNA, optionally wherein the bacterium further comprises a binary vector comprising a nucleic acid encoding the Cas nuclease.

68. The method of any one of claims 1-67, wherein the meristem cell is in a shoot apical meristem or an axillary meristem.

69. The method of any one of claims 1-67, wherein the editing of the genomic target results in the increased expression of a gene of interest in the soybean plant, wherein the genomic target inhibits the gene of interest when expressed in a control plant.

70. The method of any one of claims 1-67, wherein the genomic target is involved in viral defense, Non-Homologous End Joining (NHEJ), Mismatch Repair (MMR), or condensing chromatin.

71. The method of any one of claims 1-68, wherein the method is preceded by delivering a gene or gene fragment to repress viral defense, inhibit Non-Homologous End Joining (NHEJ), inhibit Mismatch Repair (MMR), arrest cells in the S or G2 phases, or suppress enzymes that condense chromatin.

72. The method of claim 71, wherein delivering the gene or gene fragment comprises Agrobacterium-mediated transformation or rub-inoculation.

73. The method of claim 71 or 72, wherein the gene or gene fragment is selected from the group consisting of double-stranded RNA-binding protein (DRB) genes, the DRB4 gene, genes for Dicer-like proteins, HEN1 (HUA ENHANCER 1) methyltransferase gene, and the SGS3 gene.

74. A viral vector system for use in soybean editing, the system comprising: i. a plant virus genome component; ii. one or more gRNA; and iii. a direct repeat and/or self-cleaving ribozyme sequence flanking the one or more gRNA.

75. A bean pod mottle virus (BPMV) viral vector system comprising: i. a BPMV genome component; ii. one or more gRNA; and iii. a direct repeat and/or self-cleaving ribozyme sequence flanking the one or more gRNA.

76. A viral vector system for use in soybean editing, the system comprising: i. a plant virus genome component; ii. one or more pegRNA; and iii. a Cas nuclease fused to a reverse transcriptase (RT).

77. A viral vector system for use in soybean editing, the system comprising: i. a plant virus genome component; ii. a guide RNA (gRNA) directed to a genomic target in soybean; and iii. a Casl2f nuclease.

78. A viral vector system for producing an edited genomic target in a soybean plant, the system comprising: d) a plant virus genome component; e) a guide RNA (gRNA) directed to the genomic target; and f) a Cas nuclease expressed in the meristem cell of the soybean plant.

79. A method of using a bean pod mottle virus (BPMV) to edit a genomic target in a soybean plant comprising: a) infecting the soybean plant with a recombinant bean pod mottle virus (BPMV) carrying a guide RNA (gRNA) directed to the genomic target; b) expressing a Cas nuclease in the plant; c) allowing sufficient time to elapse for the virus to infect meristem cells in the soybean plant; and thereby using the BPMV to edit the genomic target, optionally wherein the gRNA is processed by a ribozyme selected from the group consisting of a hammerhead ribozyme, a hepatitis delta virus (HDV) ribozyme, a Csy4, and a tRNA-derived sequence.

80. The method of any one of claims 1-73 and 79, the method further comprising screening the soybean plant for successful editing of the genomic target, said screening comprising: a) visually assessing the soybean plant for at least one desired phenotype; and/or b) sequencing nucleic acid of cells produced by the meristem cell after delivery of the BPMV vector.

81. A method for producing a soybean meristem cell having an edited genomic target, the method comprising: a) delivering a viral vector carrying a gRNA to a soybean meristem cell, wherein the soybean meristem cell comprises a Cas nuclease; b) allowing the gRNA and the Cas nuclease to modify the soybean meristem cell; and c) thereby producing the soybean meristem cell having the edited genomic target.

82. A method of editing a genomic target in a soybean plant scion comprising: grafting the scion onto a rootstock comprising a Cas nuclease, wherein the rootstock comprises nucleic acid encoding the Cas nuclease fused to a meristem transport segment (MTS); and delivering a guide RNA for the Cas nuclease to the scion by virus-mediated delivery, optionally wherein the scion comprises a leaf, a shoot, a stem, or other vegetative tissue.

83. The method of claim 82, the method further comprising transforming the rootstock with nucleic acid encoding the Cas nuclease prior to grafting.

84. The method of claim 82 or 83, further comprising retrieving a progeny of the scion, wherein the progeny comprises the edited genomic target.

85. A soybean plant produced by growing the seed of any one of claims 2-73, wherein the produced soybean plant comprises the edited genomic target.

86. A soybean seed produced by the method of any one of claims 1-73, wherein the produced soybean seed comprises the edited genomic target.

87. A soybean meristem cell produced by the method of any one of claims 1-73, wherein the soybean meristem cell comprises the edited genomic target.

88. A kit comprising the viral vector system of any one of claims 74-78 and an instruction manual for using the kit.