Genome editing in plants

Abstract

Compositions for genome editing and site-directed integration in plants are provided, comprising microprojectile particles coated with, treated with, or applied with recombinant DNA constructs comprising sequences encoding one or more genome editing reagents for delivery to mature embryonic explants from dry seeds. Methods for genome editing and site-directed integration in at least one cell of a plant using the disclosed compositions are also provided, as well as plants, plant parts, and seeds containing an edited genome or site-directed integration produced by the disclosed methods.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 676,228, filed May 24, 2018, the entire disclosure of which is incorporated herein by reference.

[0002] Incorporation of Sequence Listing The sequence listing measured as 4 kilobytes in the Microsoft Windows operating system and contained in the file named "MONS444WO_ST25.txt" generated on May 23, 2019, is electronically filed with this specification and is incorporated herein by reference.

[0003] Technical Field The present disclosure relates to compositions for genome editing in plants by DNA molecules encoding genome editing reagents, and methods of using them.

Background Art

[0004] Background Accurate genome editing technologies are powerful tools for manipulating gene expression and function and are expected to have the potential to improve agriculture. There is a continuing need in the art for the development of novel compositions and methods that can be used for effective and efficient editing of plant genomes.

Summary of the Invention

[0005] Summary of the Invention This disclosure provides a method for editing a plant genome, comprising delivering a recombinant DNA construct to a mature plant embryo explant, the construct comprising a sequence encoding a site-specific nuclease, the sequence operably linked to a promoter expressible in the plant, and regenerating a plant from the mature plant embryo explant, wherein the regenerated plant includes editing or site-directed integration at or near the target site of the site-specific nuclease in the genome of at least one cell of the regenerated plant. In certain embodiments, the recombinant DNA construct is delivered to the mature plant embryo explant via bacterial-mediated transformation. In other embodiments, the recombinant DNA construct is delivered to the mature plant embryo explant via Agrobacterium-mediated transformation. In some embodiments, a T-DNA transformation vector containing the recombinant DNA construct is delivered to the mature plant embryo explant.

[0006] In additional embodiments, recombinant DNA constructs are delivered to mature plant embryo explants via particle impact. In some embodiments, particles coated with or treated with recombinant DNA constructs are delivered to mature plant embryo explants via particle impact. In certain embodiments, the particles are tungsten particles, platinum particles, or gold particles. In other embodiments, the particles have a size between about 0.5 μm and about 1.5 μm. In yet another embodiment, the particles have a size of about 0.6 μm, about 0.7 μm, or about 1.3 μm. In some embodiments, multiple particles coated with or treated with recombinant DNA molecules are delivered to mature plant embryo explants via particle impact. For a particular purpose, the amount of particles delivered to the explant is between approximately 50 μg and approximately 5000 μg, or between approximately 50 μg and approximately 5000 μg, or between approximately 50 μg and approximately 2000 μg, or between approximately 50 μg and approximately 1000 μg, or between approximately 50 μg and approximately 500 μg, or between approximately 100 μg and approximately 500 μg.

[0007] In certain embodiments, the method further includes identifying regenerated plants having at least one cell containing editing or site-directed integration at or near the target site of a site-specific nuclease. In some embodiments, the identification step includes identifying regenerated plants having editing or site-directed integration based on phenotype or trait. In other embodiments, the identification step includes identifying regenerated plants having editing or site-directed integration based on a molecular assay.

[0008] In further embodiments, the site-specific nuclease is a guided nuclease (such as a CRISPR-related protein). In other embodiments, the particles are further coated with or applied with guide nucleic acids. In some embodiments, the guided nucleases are Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Csn1, Csx12, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, C The nautilus is mr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, CasZ, or an Argonaut protein, or a homolog or modified version thereof. In yet another embodiment, the nautilus is the Cas9 protein. In yet another embodiment, the Cas9 protein is from Streptococcus pyogenes. In an additional embodiment, the nautilus is the Cpf1 protein. In a particular embodiment, the site-specific nautilus is not the nautilus. In some embodiments, site-specific nucleases are meganucleases, zinc finger nucleases (ZFNs), recombinases, transposases, or transcription activator-like effector nucleases (TALENs).

[0009] In certain embodiments, the delivery step further includes delivering a second recombinant DNA construct or molecule to a mature plant embryo explant. In some embodiments, particles are further coated with or further applied with the second recombinant DNA construct or molecule. In additional embodiments, the second recombinant DNA construct or molecule is a donor template. In other embodiments, the donor template includes a homologous sequence containing a mutation for introducing a mutation into the plant genome via template-mediated repair at or near the target site of a site-specific nuclease. In yet another embodiment, the donor template includes an insertion sequence and at least one homologous sequence for incorporating an insertion sequence into the plant genome at or near the target site of a site-specific nuclease.

[0010] In certain embodiments, the insertion sequence comprises a transgene containing a coding sequence or a transcribed DNA sequence operably linked to a promoter expressible in a plant. In certain embodiments, the transgene contains a gene of interest. In some embodiments, the transgene contains a protein coding sequence. In other embodiments, the transgene contains a transcribed DNA sequence encoding a non-coding RNA molecule. In yet another embodiment, the transgene contains a marker gene. In further embodiments, the second recombinant DNA molecule contains a marker gene. In certain embodiments, the marker gene is a selectable marker gene. In some embodiments, the selectable marker gene includes an adenylyltransferase (aadA) gene, a neomycin phosphotransferase (nptII) gene, a hygromycin phosphotransferase (hpt, hph, or aph IV), a 5-enoylpyruvinylshikimic acid-3-phosphate synthase (EPSPS) gene, or a dicamba monooxygenase (DMO) gene, or a bialaphos resistance (bar) or phosphinotricin N-acetyltransferase (pat) gene. In another embodiment, the selectable marker gene includes the adenylyltransferase (aadA) gene. In an additional embodiment, the marker gene is a screenable marker gene. In various embodiments, the screenable marker gene includes the green fluorescent protein (GFP) gene or the β-glucuronidase (GUS) gene.

[0011] In further embodiments, the second recombinant DNA construct or molecule comprises a donor template region and a transgene containing a coding sequence or a transgenic DNA sequence, the transgene located outside the donor template region of the second recombinant DNA construct or molecule. In some embodiments, the second recombinant DNA construct or molecule comprises a transgenic DNA sequence encoding a guide nucleic acid, the transgenic DNA sequence being operably linked to a promoter expressible in a plant. In certain embodiments, the recombinant DNA construct further comprises a marker gene. In some embodiments, the marker gene is a selectable marker gene. In various embodiments, the selectable marker gene includes the adenylyltransferase (aadA) gene, the neomycin phosphotransferase (nptII) gene, the hygromycin phosphotransferase (hpt, hph, or aph IV) gene, the 5-enoylpyruvinylshikimic acid-3-phosphate synthase (EPSPS) gene, or the dicamba monooxygenase (DMO) gene, or the bialaphos resistance (bar) or phosphinotricin N-acetyltransferase (pat) gene. In another embodiment, the selectable marker gene includes the adenylyltransferase (aadA) gene. In yet another embodiment, the marker gene is a screenable marker gene. In yet another embodiment, the screenable marker gene includes the green fluorescent protein (GFP) gene or the β-glucuronidase (GUS) gene.

[0012] In some embodiments, the recombinant DNA construct further comprises a transcribed DNA sequence encoding a guide nucleic acid, the transcribed DNA sequence being operably linked to a promoter expressible in a second plant. In other embodiments, the recombinant DNA construct further comprises a donor template region. In certain embodiments, the donor template region comprises a homologous sequence containing a mutation for introducing a mutation into the plant genome via template-mediated repair at or near the target site of a site-specific nuclease. In yet another embodiment, the donor template region comprises an insertion sequence and at least one homologous sequence for incorporating the insertion sequence into the plant genome at or near the target site of a site-specific nuclease. In yet another embodiment, the insertion sequence comprises a transgene comprising a coding sequence or transcribed DNA sequence operably linked to a promoter expressible in a plant. In additional embodiments, the transgene comprises a gene of interest. In certain embodiments, the transgene comprises a protein coding sequence. In some embodiments, the transgene comprises a transcribed DNA sequence encoding a non-coding RNA molecule. In certain embodiments, the transgene comprises a marker gene.

[0013] In additional embodiments, the method further comprises selecting regenerated plants having a marker gene, and the marker gene being co-delivered with a recombinant DNA molecule. In some embodiments, the marker gene is a selectable marker gene. In other embodiments, the selection step comprises treating mature embryonic explants, or bud and / or root cultures, or small plants regenerated therefrom, with a selective agent. In certain embodiments, the selectable marker gene is the adenylyltransferase (aadA) gene.

[0014] Multiple embodiments relate to a method for editing the genome of a plant, comprising: a) delivering a recombinant DNA construct to a mature plant embryo explant, the construct comprising a sequence encoding one or more genome editing reagents, the sequence operably linked to a promoter expressible in the plant; and b) regenerating a plant from the mature plant embryo explant, wherein the regenerated plant contains editing or site-directed integration at or near a target site in the genome of at least one cell of the regenerated plant. In some embodiments, the method further comprises identifying a regenerated plant having at least one cell containing editing or site-directed integration at or near a target site. In some embodiments, the method further comprises selecting a regenerated plant having a marker gene, the marker gene being co-delivered with the recombinant DNA molecule. In some embodiments, the recombinant DNA construct is delivered to the mature plant embryo explant via bacterial-mediated transformation. In some embodiments, the recombinant DNA construct is delivered to the mature plant embryo explant via Agrobacterium-mediated transformation. In some embodiments, the recombinant DNA construct is delivered to the mature plant embryo explant in a T-DNA transformation vector. In some embodiments, recombinant DNA constructs are delivered to mature plant embryo explants via particle impact. In some embodiments, the particles are tungsten particles, platinum particles, or gold particles. In some embodiments, the particles have sizes of about 0.5 μm, about 0.6 μm, about 0.7 μm, about 0.8 μm, about 0.9 μm, about 1 μm, 1.1 μm, about 1.2 μm, about 1.3 μm, about 1.4 μm, or 1.5 μm. In some embodiments, the particles are further coated with or further applied a second recombinant DNA construct or molecule. In some embodiments, the second recombinant DNA construct or molecule is a donor template.

[0015] In certain embodiments, the plant is a dicotyledonous plant. In certain embodiments, the plant is a soybean plant. In other embodiments, the plant is a monocotyledonous plant. In additional embodiments, the mature embryo explant comprises, prior to the delivery step, one or more of the following: (i) a guide nucleic acid, (ii) a polynucleotide containing a transgene or marker gene, (iii) a polynucleotide containing a non-coding RNA molecule or a transgene encoding the guide nucleic acid, and / or (iv) a donor template. In some embodiments, the mature embryo explant is a dehydrated explant. In other embodiments, the mature embryo explant is a wet embryo explant, a dehydrated wet embryo explant, or a wet excised embryo explant. In yet another embodiment, the mature embryo explant has a moisture content in the range of about 3% to about 25%. In yet another embodiment, the mature embryo explant is excised from a plant seed having a moisture content in the range of about 3% to about 25%. In a further embodiment, the delivery step includes delivering a DNA molecule or vector containing a recombinant DNA construct and a second recombinant DNA construct to a mature plant embryo explant.

[0016] Brief explanation of the drawing The following drawings form part of this specification and are included to further illustrate certain aspects of the disclosure. This disclosure may be better understood by reference to one or more of these drawings in conjunction with the detailed descriptions of the specific embodiments presented herein. [Brief explanation of the drawing]

[0017] [Figure 1A] This diagram illustrates the two PDS gene loci in soybeans, located on chromosome 11 (Ch11) and chromosome 18 (Ch18), along with the target site locations for the three guide RNAs shown.

[0018] [Figure 1B]This diagram illustrates the NotI / I-CeuI restriction fragment excised from a vector containing expression cassettes for the gus marker gene, aadA marker gene, Cas9 nuclease, and guide RNA, which is used for particle impact on the explant to generate genome editing at a target site within the PDS gene locus.

[0019] [Figure 1C] This diagram illustrates the different possible orientations for the insertion of a complete NotI / I-CeuI restriction fragment into the PDS locus, along with the binding sites for the PCR primers (3965, 3966, 2438, and 4005) shown for each orientation.

[0020] [Figure 2] This is an image of a gel showing PCR products generated using different primer pairs (3965 and 4005 or 3966 and 4005), and showing two site-directed inclusion (SDI) events (event #20 and event #48) in the edited sample tested. [Modes for carrying out the invention]

[0021] Detailed explanation Gene function analysis and crop improvement using genome editing technologies are very promising in agricultural improvement. Genome editing reagents are delivered in the form of DNA into plant cells that can be cultured, but delivering the DNA encoding the genome editing reagent into mature plant embryo explants to regenerate or generate edited plants has not been described. Plant cells and tissues transformed with the DNA encoding the genome editing reagent may have limited regenerative ability, and many plant genetic resources may not be suitable for these culture methods. In fact, many crop plants and cultivars cannot effectively form callus tissue, suspension cultures, or protoplasts. Thus, existing genome editing methods can be species- and genotype-dependent depending on the type of explant and culture requirements, and in many cases are limited to less commercially important genetic resources and cultivars of agriculturally economically important crops, which require multiple rounds of backcrossing with a promising donor line to introgress genome editing or site-directed integration into the desired genetic background.

[0022] This disclosure overcomes a gap in the art by providing a method for delivering a genome editing reagent (such as a nuclease protein or guide nucleic acid) into a mature embryo explant or a desiccated embryo explant (DEE) as DNA encoding the genome editing reagent, which can be delivered via bacterial-mediated transformation or coated onto particles for microparticle gun delivery to the explant. A “desiccated explant” is a mature embryo explant harvested or excised from a mature, dried seed. Plant seeds dry naturally during their maturation process. Further, as described below, other types of explants may be harvested or excised from mature seeds depending on their processing (such as after wetting, absorption, etc. of the dried seed), and the explants may be wetting, absorbed, etc. after excision from the dried seed. Editing or site-directed integration can be achieved by delivering DNA encoding a genome editing reagent (such as a nuclease protein or guide nucleic acid) to one or more cells of meristem-related tissue (such as embryonic meristem tissue) without a prior callus formation step. By avoiding the need for a callus phase prior to the delivery of genome editing reagents into one or more cells of targeted explants according to this method, the genotype and species dependence of conventional methods is reduced or eliminated, and effective delivery of DNA encoding genome editing reagents (such as nuclease proteins or guide nucleic acids) into those targeted explants can be achieved. Therefore, the method disclosed herein for genome editing reagent delivery can be directly applied to a variety of plant genetic resources (including promising genetic resource lines of agroeconomically important crop species), which may enable the direct regeneration of plants of desired genetic resources containing site-directed integration of targeted edits or insertion sequences or transgenes.

[0023] Desired edited or arrayed or site-directed integration of transgenes in the dried excised explants is generated according to the genome editing method of the present disclosure, which can be made to normally occur rather to adult R0 plants containing the desired editing or site-directed integration by only a few culture steps and / or regeneration steps. Such R0 plants can be generated or regenerated from the explants without the need for embryo development culture or callus culture. The targeted editing or site-directed integration in the R0 plants produced by the method of the present disclosure is further transmitted into the germ cell line, and in addition to the genome-edited R1 seeds and plants, subsequent generations of seeds and plants with the desired editing or site-directed integration can be produced.

[0024] The ability to generate genome-edited R0 plants without large-scale culture of desiccated explants prior to the introduction of genome editing reagents (such as nuclease proteins or guide nucleic acids) enables the method of this disclosure to be carried out more rapidly and efficiently, thus enabling its potential for implementation in the large-scale commercial production of genome-edited crop plants. Desiccated explants can be harvested from seeds and used almost directly as targets for genome editing or site-directed integration. According to some embodiments, desiccated explants can be harvested from mature dried seeds and possibly used as targets for editing with only minimal wetting, hydration, or pre-culture steps. Thus, desiccated explants from storable dried seeds can be conveniently utilized as targets for genome editing or site-directed integration of insertion sequences or transgenes. As an alternative to desiccated explants, "moist" embryo explants, or "dried moist" embryo explants (e.g., primed embryo explants or germinated embryo explants), can be used as targets for genome editing. Such “moist” embryonic explants are dry-excised explants that undergo moistening, hydration, absorption, or other minimal culture steps before receiving editing enzymes. Similarly, “moist-excised” explants from absorbed or hydrated seeds can also be used as targets. “Moist” embryonic explants are hydrated or absorbed after excision from the seed, while “moist-excised” embryonic explants are excised from seeds that have already been hydrated or absorbed.

[0025] I. Method of Transformation Embodiments of the present disclosure provide a method for editing the genome of a desiccated excised explant (DEE) from a plant's dried seed, comprising introducing DNA encoding a genome editing reagent (such as a nuclease protein or guide nucleic acid), which may be provided with a donor template, into at least one cell of the explant for transformation via particle-mediated microparticle gun or bacteria-mediated (e.g., Agrobacterium-mediated) delivery, thereby producing genome editing or site-directed integration in at least one cell of the explant. The method of the present disclosure may be carried out by targeting a desiccated excised embryo explant from harvested seeds without large-scale culture of the explant prior to delivery of DNA encoding one or more genome editing reagents. Such an explant may be excised from a storable dried seed or may be a “moist” embryo explant, a “dried moist” embryo explant, or a “moist excised” embryo explant.

[0026] According to some embodiments, dried explants excised from plant seeds can be optionally pre-cultured in an aqueous medium for a limited time before delivering DNA encoding a genome editing reagent (such as a nuclease protein or guide nucleic acid) to the explant. Such a pre-culture medium may contain various salts (multiple) (e.g., MS basic salt, B5 salt, etc.) and other components (various osmoregulators (multiple), sugars (multiple), antimicrobial agents (multiple), etc.). The pre-culture medium may be solid or liquid and may further contain one or more plant growth regulators or plant hormones (such as auxins (multiple), cytokinins (multiple), etc.). According to some embodiments, multiple explants can be pre-cultured together in the same medium or container. For example, a range of 2 to 100 explants (approximately 25, 50, 75, or 100 explants, etc.) may be plated on or in the same pre-culture medium, but a larger number of explants may be pre-cultured together depending on the type of explant, the size of the container, the dish, etc. According to some embodiments, the pre-culture medium may contain auxin (2,4-D, indoleacetic acid (IAA), dicamba, 1-naphthaleneacetic acid (NAA), etc.) and cytokinin or similar growth regulators (thidiazurone (TDZ), 6-benzylaminopurine (BAP), zeatin, or zeatin riboside, etc.). Such pre-culture or pre-culture step may promote the ability of the explants to edit and / or regenerate. The relative amounts of auxin and cytokinin (or similar growth regulators) in the pre-culture medium can be controlled or predetermined to improve the success of editing and / or regeneration while avoiding callus formation from the explant (possibly over an extended period). According to some embodiments, the pre-culture medium may contain both auxin (e.g., 2,4-D) and cytokinin (e.g., TDZ). For example, the concentration of cytokinin (e.g., TDZ) in the pre-culture medium (if present) may be in the range of zero (0) to about 5 ppm (about 0.3 to about 4 parts per million (ppm), about 0.5 to about 3 ppm, about 1 to about 2, etc.) or within other intermediate ranges.In a particular manner, the concentration of cytokinin in the pre-culture medium may be 0, about 0.1 ppm, about 0.2 ppm, about 0.3 ppm, about 0.4 ppm, about 0.5 ppm, about 0.6 ppm, about 0.7 ppm, about 0.8 ppm, about 0.9 ppm, about 1.0 ppm, about 1.25 ppm, about 1.5 ppm, about 1.75 ppm, about 2 ppm, about 2.5 ppm, about 3 ppm, about 3.5 ppm, about 4 ppm, about 4.5 ppm, or about 5 ppm. In the case of TDZ, the concentration may preferably be less than 2 ppm, or in the range of about 0.7 to about 1.3 ppm, or about 0.5 to about 1 ppm, or about 0.3 ppm or about 1.5 ppm. In a particular manner, the concentration of TDZ is approximately 0.1 ppm, 0.2 ppm, 0.3 ppm, 0.4 ppm, 0.5 ppm, 0.6 ppm, 0.7 ppm, 0.8 ppm, 0.9 ppm, 1.0 ppm, 1.1 ppm, 1.2 ppm, 1.3 ppm, 1.4 ppm, 1.5 ppm, 1.6 ppm, 1.7 ppm, 1.8 ppm, or 1.9 ppm. The concentration of auxin (2,4-D, etc.) may be in the range of zero (0) to approximately 2 ppm, or approximately 0.1 ppm to approximately 1 ppm, or approximately 0.1 ppm to approximately 0.5 ppm, or within other intermediate ranges. In a particular manner, the auxin concentration is approximately 0.1 ppm, 0.2 ppm, 0.3 ppm, 0.4 ppm, 0.5 ppm, 0.6 ppm, 0.7 ppm, 0.8 ppm, 0.9 ppm, 1.0 ppm, 1.1 ppm, 1.2 ppm, 1.3 ppm, 1.4 ppm, 1.5 ppm, 1.6 ppm, 1.7 ppm, 1.8 ppm, or 1.9 ppm.

[0027] The duration of the pre-culture step may vary, depending in part on the temperature of the pre-culture medium and / or the surrounding temperature of the explant. Generally, the duration of the pre-culture step can also be controlled and limited to a range of about 1 or 2 hours to about 5 days (e.g., about 12 hours to about 60 hours or about 12 hours to about 48 hours), or to other periods within that range. Limiting the amount of time for the pre-culture step can also avoid callus formation, despite the presence of plant growth regulators. An optimal pre-culture duration can also improve the frequency of plant regeneration. During the pre-culture step, the explant may be maintained in the same medium or transferred to fresh medium one or more times. Optional lighting and / or temperature conditions for the pre-culture step can also be controlled. For example, the explant may be exposed to a 16 / 8 hour photoperiod during the pre-culture step, or possibly to various other light-dark cycles or periods. Alternatively, the pre-culture step may be performed in darkness or low light conditions. The temperature of the pre-culture medium and environment of the explants also varies between approximately 18°C ​​and 35°C, or between approximately 25°C and 30°C, or around 28°C, and may include all intermediate ranges and values.

[0028] According to some embodiments, and regardless of whether a pre-culture step is performed, excised explants for transformation may be optionally exposed to a hydration or absorption medium for a limited time prior to pre-culture and / or exposure to genome editing reagents. Such hydration or absorption step may make explants from dried seeds or dried seeds more suitable for editing or site-directed integration. In fact, the hydration or absorption step may be performed without an isolated pre-culture step prior to transformation. The hydration medium may consist of water alone or may further contain one or more known osmoregulators (multiple) (sugars (multiple) (e.g., sucrose), polyethylene glycol (PEG), etc.). For example, the hydration medium may contain about 10% sucrose and / or about 20% PEG. Although not bound by any theory, osmoregulators may regulate or slow the hydration rate of the explants. Other components may also be included in the hydration medium (various salts, etc.). The duration for the hydration step will generally be short (approximately 2 minutes to 12 hours, or approximately 20 minutes to 6 hours, or approximately 30 minutes to 2 hours, or approximately 1 hour, etc.). The hydration or absorption step may be short enough so that germination or at least any observable germination or developmental change does not occur in the explant. Alternatively, the embryonic explant may be primed for germination, or possibly germinated, before the delivery of the genome editing reagent. For example, the embryonic explant may be primed for germination by wetting, and then the explant may be dried (to produce a “dried wet” embryonic explant) to stop germination. Furthermore, “wet excised” embryos (embryonic seeds or embryonic explants excised from wet seeds) may also be used as targets for transformation. Various rinsing steps may also be performed before, after, and / or in between any of the hydration steps and / or pre-culture steps.

[0029] According to some embodiments, hydration steps and / or pre-culture steps are included prior to transformation to improve editing, particularly for dry (or dehydrated) explants (such as those taken from mature and / or dry (or dehydrated) seeds), although either or both of these steps may depend at will on the water content and / or the type of explant used as the target for editing. However, since "moist" embryonic explants or "moist excised" embryonic explants may already have a sufficient level of hydration or water content, especially when used as the target, the hydration steps and / or pre-culture steps may be optional, not included, or not performed.

[0030] Whether or not hydration steps and / or pre-culture steps are performed, the explant can be transformed with a DNA molecule encoding a genome editing reagent (such as a nuclease protein or guide nucleic acid), which can be coated onto particles for microparticle gun delivery or incorporated into a vector (e.g., a T-DNA vector) for transformation, to produce an explant having at least one genome-edited cell. Following the delivery or transformation of the genome-editing reagent-encoding DNA, the explant can then be grown, developed, regenerated, etc., into a plant under selective pressure to select for the growth and development of the genome-edited cell(s) of the explant. In certain embodiments, one or more genome-editing reagent-encoding DNA can be co-transformed or co-delivered with a selectable marker gene so that the survival, growth, and development of the genome-edited cell may be advantageous in the presence of the corresponding selective agent. According to some embodiments, one or more genome-editing reagent-encoding DNA can be co-transformed or co-delivered into the genome of a plant cell with a donor template molecule for site-directed integration of an insertion sequence or transgene (e.g., a gene of interest). The donor template molecule for site-directed integration may further include selectable marker genes that can be used as a base for selection. According to some embodiments, DNA encoding one or more genome editing reagents may be co-transformed or co-delivered together with a guide nucleic acid, or DNA encoding a guide nucleic acid. According to some embodiments, the DNA encoding one or more genome editing reagents that are transformed or delivered to the explant may also include one or more of the donor template sequences and / or selectable marker genes.

[0031] According to certain embodiments of this disclosure, a transformation vector incorporating DNA encoding one or more genome editing reagents, or particles having, coated with, or applied to such DNA on their surface, are introduced into at least one cell of a target explant via explant transformation or particle-mediated impact. Such particle-mediated impact can be carried out using any suitable particle gun device known in the art (e.g., helium particle gun, electric particle gun, etc.). Prior to impact, the particles may be loaded with, applied to, or coated with a copy of a DNA construct or molecule encoding one or more genome editing reagents, and optionally with guide RNA, marker genes, and / or donor templates. The DNA construct or molecule encoding one or more genome editing reagents may include sequences encoding guide RNA, marker genes, and / or donor templates. The particles themselves may be any suitable type of particle or bead known in the art, such as gold beads or tungsten beads. Blasting conditions for particle guns are well known in the art, and various conventional screens, rupture discs, etc., can be used for helium particle guns, etc. Electric guns can offer several advantages by reducing the amount of time required for transformation and by using fewer consumables in the process.

[0032] For particle impact, dried embryo explants can be plated onto a target medium or substrate that can hold the explant in place and orient it appropriately for spraying. Such a target medium or substrate may contain, for example, a gelling agent (such as agar and carboxymethylcellulose (CMC)) to control its viscosity. Plating the explant in a liquid (such as hydrated medium, pre-culture medium, or rinse medium) can facilitate the spreading and positioning of the explant. Explants targeted for particle impact according to a particular embodiment may be positioned so that the tissue related to the meristem of the explant preferentially receives the spraying particles. For example, explants can be placed on a surface with their meristems facing upwards to preferentially receive the coated particles during impact. Each explant can be sprayed with coated particles under varying pressures, forces, and / or in a single or multiple pass.

[0033] According to embodiments of this disclosure in which a selectable marker gene is co-delivered with DNA sequences encoding one or more genome editing reagents, the targeted explant can be cultured on (or in) a selective medium (or series of selective media) after shock or transformation to enable or select the cells and tissues of the explant containing the selectable marker gene to regenerate or develop into a plant or part of a plant (such as a root and / or bud). The selectable marker gene is co-delivered with DNA sequences encoding one or more genome editing reagents to select cells that can express one or more genome editing reagents together with the selectable marker gene. Generally, the selective medium will contain a selective agent and, based on the expression of the selectable marker gene delivered to at least one cell of the explant, will bias or support the survival, proliferation, mitotic growth, and / or development of the cells of the explant (when expressed in recipient cells and their progeny cells, the selectable marker gene provides resistance to the selective agent). According to some embodiments, however, the impacted or transformed explants do not need to be subjected to selective pressure, and the resulting or regenerated plants can ultimately be screened for the presence of editing or mutations at the target site.

[0034] According to some embodiments, however, the explant may be optionally cultured on (or in) a first resting medium of a first post-culture lacking the selective agent for a first period immediately after transformation or impact of the target explant, allowing the explant to recover and / or begin expressing a selectable marker gene. Such a resting step may be a period within the range of about 1 to 24 hours, or about 6 to 18 hours, or about 10 to 15 hours (e.g., about 12 hours or overnight). Recovery of the edited plant may be improved by providing a non-selective period for recovery (e.g., culture on resting medium), but if selection is initiated too late (e.g., more than 18 to 24 hours after impact), the frequency of recovering edited plants may decrease. Each of the post-culture medium, selection medium, or resting medium contains standard plant tissue culture medium components (salts, sugars, plant growth regulators, etc.), and cultivation on these media can be carried out at standard or modified temperatures (e.g., 28°C) and lighting conditions (e.g., a 16 / 8-hour photoperiod). However, the first post-culture or resting step may be included before selection or omitted, depending on the editing frequency and selection scheme (specific selectable marker genes and selection agents used, etc.).

[0035] Following any initial recovery and culture of the explant on a first non-selective resting medium, the explant may optionally undergo a fermentation step. According to these embodiments, the explant may be exposed to, placed on, or in a second post-culture medium or fermentation medium containing an osmotic regulator (e.g., polyethylene glycol (PEG)) and / or a calcium-containing salt compound (e.g., calcium nitrate [Ca(NO3)2]). For example, the concentration of calcium nitrate may be about 0.1 M and the concentration of PEG may be about 20%, although their concentrations may vary. This fermentation medium may also lack selective agents. Exposure of the impacted explant to the fermentation medium may function to further drive coated particles and / or DNA and marker gene constructs (if used) encoding one or more genome editing reagents into the explant cells. The explants are placed in or on the fermentation medium for only a short period (ranging from approximately 30 minutes to approximately 2 hours, or approximately 1 hour, etc.), followed by a rinse step(s), after which there may be any further culture or selection steps.

[0036] As described above, impacted or transformed explants can be brought into contact with one or more selective media containing selective agents to bias the survival, proliferation, mitotic growth, and / or development of cells expressing the selectable marker gene construct used for co-transformation. Selectable marker genes will generally be paired with the selective agent used for selection so that the selectable marker gene confers resistance to selection by the selective agent. For example, the selectable marker gene could be the adenylyltransferase gene (aadA) that confers resistance to spectinomycin or streptomycin as the selective agent.

[0037] A plant-selectable marker gene or transgene may include any gene that confers resistance to the corresponding selective agent, enabling plant cells transformed with the plant-selectable marker transgene to withstand the selective pressure imposed by the selective agent. As a result, cells in explants receiving the selectable marker gene are favored for selective proliferation, mitosis, and development. While plant-selectable marker genes are generally used to confer resistance to selective agents, additional screenable marker or reporter genes may also be used. Examples of such screenable marker or reporter genes include β-glucuronidase (GUS; e.g., as described in U.S. Patent No. 5,599,670) or green fluorescent protein and its variants (GFP, as described in U.S. Patents No. 5,491,084 and 6,146,826). Various screenable markers or reporter genes detectable in plants, plant parts, or plant cells are known in the art, including luciferases, other non-GFP fluorescent proteins, and genes that confer a detectable phenotype in plants, plant parts, or seeds (e.g., phytonene synthase). Additional examples of screenable markers include secretory markers (e.g., opine synthase genes) whose expression triggers the secretion of the molecule(s) whose expression can be detected as a means of identifying transformed cells.

[0038] Plant-selectable marker genes may include genes encoding proteins that provide or confer resistance or tolerance to herbicides (such as glyphosate and glufosinate). Useful plant-selectable marker genes are known in the art and may include those encoding proteins that confer resistance or tolerance to streptomycin or spectinomycin (e.g., adenylyltransferase, aadA, or spec / strep), kanamycin (e.g., neomycin phosphotransferase or nptII), hygromycin B (e.g., hygromycin phosphotransferase, hpt, hph, or aph IV), gentamicin (e.g., aac3 and aacC4), and chloramphenicol (e.g., chloramphenicol acetyltransferase or CAT).Additional examples of known plant selectable marker genes encoding proteins that confer herbicide resistance or tolerance include, for example, a transcriptable DNA molecule encoding 5-enoylpyruvinylshikimic acid-3-phosphate (EPSP) synthase (EPSP for glyphosate resistance; e.g., described in U.S. Patent Nos. 5,627,061; 5,633,435; 6,040,497; and 5,094,945); a transcriptable DNA molecule encoding glyphosate oxidoreductase and glyphosate-N-acetyltransferase (GOX; e.g., described in U.S. Patent No. 5,463,175; GAT, described in U.S. Patent Publication No. 2003 / 0083480); and a transcriptable DNA molecule encoding phytoendesaturase (crtI; e.g., for norflurazone resistance, Misawa, et al., Plant Examples include the dicamba monooxygenase (DMO) gene encoding the dicamba monooxygenase (DMO) gene for dicamba resistance (e.g., U.S. Patent Applications No. 2003 / 0115626 and 2003 / 0135879; and Behrens et al., Science 316(5828):1185 2007); and the bialaphos resistance (bar) or phosphinotricin N-acetyltransferase (pat) gene for glufosinate and bialaphos resistance (e.g., DeBlock et al., EMBO Journal, 6:2513-2519 (1987)).

[0039] To perform the selection step(s), the explant may be brought into contact with or placed on (or in) one or more selective media containing selective agents. In addition to the application of selective pressure, the selective media may simultaneously provide regeneration or development of buds, roots and / or whole plants from the impacted explant. Alternatively, a regeneration medium may be used for the development or regeneration of one or more buds and / or roots without the presence of selective agents. The regeneration medium and / or selective media may contain various standard plant tissue culture components (salts (e.g., MS or B5 salts), sugars(s), etc.). The regeneration medium and / or selective media may optionally contain plant growth regulators(s) (auxin and / or cytokinins, etc.) that may enhance or assist the development, elongation or regeneration of buds and / or roots (and ultimately the whole plant). The regeneration step(s) and / or selection step(s) may be performed within a range of standard or varied temperatures (e.g., 28°C) and lighting conditions (e.g., 16 / 8 photoperiod). Such development of genome-edited R0 plants from impacted explants on selective media may largely resemble the normal processes of germination and plant development, although some rearrangement of the meristem may occur in response to selective pressure, potentially forming buds and / or roots, as well as other parts of the adult plant. Importantly, not only is the callus phase avoided before the impact or transformation step, but the explant may also further develop or regenerate into a genome-edited R0 plant after transformation without forming embryogenetic callus from the explant.

[0040] According to embodiments of the present disclosure, an explant may be cultured in a first selective medium (or a series of selective media) until a green bud is formed, after which it may be harvested or cut and transferred to a new selective medium. The transfer or subculturing process may be repeated once or multiple times (e.g., two, three, four, or five times) to provide multiple rounds of transfer, subculturing, and / or selection. Multiple rounds of transfer, subculturing, and / or selection of buds from explants under selective pressure are thought to be able to expand or increase the number, proportion, and / or ubiquity of genome-edited cells throughout the subsequent developing or regenerated genome-edited R0 plant.

[0041] According to some embodiments, a regeneration medium (which may also be a selective medium, such as one or more of the selective media described above) also functions as a rooting medium to induce or enable the formation and development of roots (or more) from transplanted or subcultured shoots (or more), which may contain one or more plant growth regulators (or more) (such as auxin and / or cytokinin). Each rooting medium (or more) is also a selective medium and may contain a selective agent in addition to one or more plant growth regulators. Rooting microplants developed or regenerated from impacted explants (via continuous transplantation or subculture under selective pressure) are ultimately transplanted into PlantCon® or other suitable containers and / or pots for the continuous development of genome-edited R0 plants, and genome-edited R1 seeds can then be harvested from those genome-edited R0 plants. Sequential subculturing (and eventual rooting) of green shoots derived from initially impacted or transformed explants under selective pressure of only a few rounds may be sufficient to form genome-edited R0 microplants that can further develop into genome-edited R1 plants and fertile plants that produce seeds. This disclosure represents a significant advance and improvement in the art by providing the production of genome-edited plants at reasonable frequencies in different plant genetic resources. In practice, the method of this disclosure avoids the need for a callus phase at any stage by preparing explants that have been desiccanted for impact or transformation, and then generating or regenerating genome-edited R0 plants from the impacted or transformed explants. In contrast to this disclosure, existing methods for genome editing are generally limited to specific explant types and plant genetic resources and cultivars that are suitable for larger-scale culture steps.

[0042] According to embodiments of this disclosure, one or more selection steps may be carried out in a single-selection medium, or more preferably in a series of selection steps or media. The amount or concentration of the selection agent in the selection medium may vary depending on the specific selection agent used. For example, the amount of spectinomycin used for the selectable marker gene aadA may be in the range of about 50 ppm to about 250 ppm, or about 100 ppm or about 150 ppm. According to some embodiments, the amount or concentration of the selection agent may remain constant throughout the selection period, or the amount or concentration of the selection agent may be stepped up or increased over the selection period. A step-by-step approach may allow more time for the transformed explant cell(s) to recover until they can achieve more steady expression of the selectable marker gene and withstand stronger selection pressures. However, expression of the selectable marker gene may be sufficient within the time of the initial selection pressure, and as a result, a step-by-step selection approach would be unnecessary. In either approach, the explants may be periodically transferred to or subcultured in fresh selective medium, or the selective medium may be periodically replaced with new selective medium. According to some embodiments, the explants may be maintained in or on each of the selective media for a period of about several days (e.g., 2 or 3 days) to several weeks (e.g., 3 to 4 weeks), or within a range of about 1 to 3 weeks, or for about 2 weeks, before being transferred to or subcultured in the next medium. According to specific embodiments involving the use of spectinomycin as the selective agent, the concentration of spectinomycin may be increased in a stepwise manner from about 50 ppm to about 500 ppm, or alternatively, the amount concentration of spectinomycin may be maintained relatively constant (e.g., at about 100 ppm, about 150 ppm, or about 200 ppm).

[0043] According to some embodiments, DNA encoding one or more genome editing reagents for genome editing can be transformed into at least one cell of a mature embryonic explant using any transformation method known in the art. Various methods for transferring genes into plant tissues are known, including high-speed microprojection, microinjection, electroporation, direct DNA uptake, and bacterial-mediated transformation. According to some embodiments, DNA encoding one or more genome editing reagents for genome editing can be transformed into at least one cell of a mature embryonic explant via bacterial-mediated transformation. Bacteria known to mediate the transformation of plant cells include numerous species of the families Rhizobiaceae and Rhizobia, which include, but are not limited to, species of the genera Agrobacterium, Sinorhizobium, Mesorhizobium, and Bradyrhizobium (see, e.g., Broothaerts et al., 2005; and U.S. Patent Applications Publications 2007 / 0271627 and 2008 / 0280361).

[0044] Bacterial delivery of DNA molecules (e.g., Agrobacterium-mediated delivery; see also U.S. Patents No. 5,563,055; No. 5,591,616; No. 5,693,512; No. 5,824,877; and No. 5,981,840) occurs in cells within the living meristem of an embryo excised from a seed (such as soybean and other crop seeds). The region relating to the meristem can be cultured in the presence of a selective agent to regenerate an R0 plant having one or more cells transformed with DNA molecules. Bud and / or root formation can occur in various culture media to regenerate a plant from one or more explant cells, which may include cells relating to the transformed meristem.

[0045] Possible selective agents include auxin-like herbicides (dicamba or 2,4-D, MCPA, etc.), glufosinate, acetolactic acid synthase inhibitors, protoporphyrinogen oxidase inhibitors and hydroxyphenyl-pirubate-dioxygenase inhibitors, neomycin, kanamycin, paramomycin, G418, aminoglycosides, spectinomycin, streptomycin, hygromycin B, bleomycin, phleomycin, sulfonamides, streptotrycin, chloramphenicol, methotrexate, 2-deoxyglucose, betainaldehyde, S-aminoethyl L-cysteine, 4-methyltryptophan, D-xylose, D-mannose, and benzyladenine-N-3-glucuronidase. Examples of selectable marker genes that provide resistance to these selective agents are also known in the art.

[0046] A variety of tissue culture media are known that, when properly supplemented, support the proliferation and development of plant tissues (including the formation of mature plants from excised meristems or embryos). These tissue culture media may be purchased as commercial preparations or custom-prepared and may be modified by those skilled in the art. Examples of such media include, but are not limited to, those described by Murashige and Skoog, (1962); Chu et al., (1975); Linsmaier and Skoog, (1965); Uchimiya and Murashige, (1962); Gamborg et al., (1968); Duncan et al., (1985); McCown and Lloyd, (1981); Nitsch and Nitsch, (1969); and Schenk and Hildebrandt, (1972), or derivatives of these media supplemented accordingly. Those skilled in the art will know that culture media and media supplements (such as nutrients and growth regulators for use in transformation and regeneration) are typically optimized for specific target crops or varieties of interest. Reagents are commercially available and can be purchased from numerous suppliers (see, for example, Sigma Chemical Co., St. Louis, Mo., and Phytotechnology Laboratories, Shawnee Mission, Kans.).

[0047] Co-culturing and subsequent steps may be carried out for 2 to 5 days under dark conditions or in an illuminated Percival incubator, for example, with a light cycle of 16 hours of light and 8 hours of dark. In one embodiment, the light intensity may be at least about 5 μE (including about 10 μE or 25 μE, and including at least about 5 μE to about 200 μE) or other illumination conditions, carried out at temperatures of approximately 23 to 25°C and up to about 35°C to allow normal plastid development.

[0048] The methods of this disclosure enable the regeneration and / or development of candidate genome-edited plants from one or more impacted or transformed explants without the need for large-scale culture, thus increasing efficiency in the identification and propagation of buds and plants containing one or more genome-edited cells and reducing the cost and effort required to produce genome-edited plants of desired varieties or genetic resources. For example, after putative transformants are identified using selection markers, the plantlets may be placed in soil or on a soil substitute (such as a rooting medium) with or without the presence of a selection agent. Budlets elongating from selected or regenerated explants can be assayed for the presence of genome editing at target sites using molecular techniques. Genome-edited R0 plants may further give rise to genome-edited R1 plants and seeds, which may also produce subsequent genome-edited offspring plants and seeds. Genome-edited R0 plants may be produced by the methods of this disclosure with little or no selective pressure, although the maintenance of selection with appropriate selection agents may be maintained over one or more culture or regeneration steps. R1 plants determined to have one or more genome edits at desired target sites are crossed with another plant, and homozygous genome-edited plants can be selected in subsequent generations to have fixed mutations or edits with respect to the genetic inheritance of genome edits or mutations in subsequent generations (without segregation of edits or mutations in the offspring plants, and with stable maintenance of homozygosity in offspring through self-crossing). As described above, the proliferation, survival, and development of genome-edited cells in R0 plants can also be selectively or preferentially achieved or supported by applying selective pressure with selective agents during the explant culture, subculturing, bud elongation, and / or rooting steps, thereby producing R0 plants with a higher proportion of genome-edited(s) or mutated(s) cells resulting from co-delivery of selectable marker genes. However, the selective pressure can be alternatively continued (e.g., periodically) after the initial culture and / or during the remaining lifespan of the R0 plant (e.g., as a topical spray, soil, or seed application).

[0049] A variety of tissue culture media are known that, when properly supplemented, support the proliferation and development of plant tissues (including the formation of mature plants from excised plant tissue). These tissue culture media may be purchased as commercial preparations or custom-prepared and may be modified by those skilled in the art. Examples of such media include Murashige and Skoog,Physiol.Plant 15:473-497,1962);Chu et al.,(Scientia Sinica 18:659-668,1975);Linsmaier and Skoog,(Physiol.Plant 18:100-127,1965);Uchimiya and Murashige,Plant Physiol.57:424-429,1976;Gamborg et al.,Exp.Cell Res.50:151-158,1968;Duncan et al.,Planta 165:322-332,1985;McCown and Lloyd,HortScience 16:453,1981;Nitsch and Nitsch Plant Examples include, but are not limited to, those described by Physiol. 44:1747-1748, 1969; and Schenk and Hildebrandt, Can. J. Bot. 50:199-204, 1972, or derivatives of these media supplemented accordingly. Those skilled in the art will know that media and media supplements (nutrients and plant growth regulators for use in transformation, selection, and regeneration, etc.) are usually optimized for specific target crops or varieties of interest. Tissue culture media may be supplemented with carbohydrates (glucose, sucrose, maltose, mannose, fructose, lactose, galactose, and / or dextrose, etc., but not limited to these) or by carbohydrate ratios. Reagents are commercially available and can be purchased from numerous suppliers (see, for example, Sigma Chemical Co., St. Louis, MO; and PhytoTechnology Laboratories, Shawnee Mission, KS).These tissue culture media can be used as resting or selective media by further addition of selective agents, and / or as regeneration media if supplemented with one or more plant growth regulators.

[0050] Embodiments of the present disclosure also provide genome-edited plants, plant parts, and seeds produced by the transformation methods of the present disclosure, which include one or more edits or mutations at or near a target site. Plant parts include, but are not limited to, fruits, seeds, endosperm, ovules, pollen, leaves, stems, and roots. In certain embodiments of the present disclosure, the plant or plant part is a seed.

[0051] II. Transformable explants The methods of the present disclosure may further include a step(s) of excising at least a portion of a plant embryo from a plant seed by any preferred manual or automated method prior to transformation, or the excision thereof. According to embodiments of the present disclosure, a preferred embryo explant further includes the meristem or tissue relating to the meristem of the embryo, or at least a portion of the meristem, or cells relating to at least one meristem of the embryo explant, for the reason that targeting cells relating to the meristem of the explant for transformation for the delivery of DNA encoding one or more genome editing reagents may improve or be necessary for the effective generation and development or regeneration of genome-edited plants. The embryo explant may lack one or more embryonic tissues (e.g., cotyledons, hypocotyls, radicles, etc.) as long as it retains at least a portion of the embryo meristem. The use of mature embryo explants excised from dried seeds is preferred according to many embodiments of the present disclosure, which may require a hydration step(s) and / or pre-culture step(s) prior to transformation.

[0052] Methods suitable for producing or excising embryonic explants from any plant seed may be used in conjunction with embodiments of the present disclosure. These methods may include a single-process or bulk process that can be automated and / or performed manually. According to many embodiments, the embryonic explant may be a mature embryonic explant (or a portion thereof) collected or excised from a dried mature plant seed. For any given species of plant, a mature seed or embryo may be defined in that it is above a certain number of days after pollination (DAP) to distinguish it from an immature seed or embryo of the same species of plant, although for a given plant species, the transition from immature to mature embryo may be gradual. Generally, as is known in the art, the transition from immature to mature embryo is accompanied by a natural process of drying or dehydration of the seed and embryo (in addition to other developmental changes).

[0053] Since drying is associated with the development or maturation of seeds and embryos, the mature seed or embryo explants used in the methods of this disclosure can also be defined in terms of their water content. Furthermore, embryo explants can be defined in terms of the water content of the seed from which they are excised. For example, the seed or embryo explants used according to this method may initially have a water content in the range of about 3% to about 25%, or about 4% to about 25%, or about 3% or 4% to about 20%, or, depending on the particular species of plant, any percentage value or range within such a broader percentage range (e.g., about 5% to about 20%, about 5% to about 15%, about 8% to about 15%, and about 8% to about 13%). In fact, plant seeds can be artificially dried or dehydrated before excision of embryo explants prior to use in embodiments of the methods of this disclosure, as long as the seeds and embryos maintain viability and are competent for plant transformation and development or regeneration. Drying of seeds may facilitate the excision and / or storage of embryonic explants from the seeds. Alternatively or additionally, seeds may be hydrated or absorbed before excision of the explants to promote embryo development, soften the embryo, reduce damage to the embryo, and / or maintain embryonic viability during the excision step. However, even if the seeds or explants are subsequently dried or dehydrated, hydration of the seeds or explants may reduce or eliminate their storability.

[0054] For further descriptions of explants and methods for excising explants from previously hydrated, primed, or germinated dried seeds, dried seeds, and / or mature seeds, see, for example, U.S. Patents 8,466,345, 8,362,317, 8,044,260, and U.S. Patent Publication 2016 / 0264983. Regardless of the type of seed used and the exact method of mechanically excising the explants from the seeds, additional steps and processes (sterilization, thinning, etc.) may also be performed to prepare and / or enrich the explants to be used for particle impact. Dried or dried explants may also be hydrated, primed, and / or germinated after their excision but before the transformation step.

[0055] The embryonic explants used in this disclosure may be removed from the seed less than one day before use in the method (e.g., about 1 to 24 hours before use, including about 2, 6, 12, 18, or 22 hours before use). According to other embodiments, however, the seeds and / or explants may be stored for longer periods, depending on the storage conditions used to maintain the viability of the seeds and / or explants, including several days, weeks, months, or possibly years before their use. The advantage and benefit of using dry, mature seeds as a source for producing or excising embryonic explants suitable for genome editing is that the dry, mature seeds and / or explants may be storable under dry conditions (not germinating, maintaining viability, and being competent for transformation during storage). Such dry storage conditions can be defined as being stored in an environment or surroundings with sufficiently low moisture or humidity so that the stored seeds and / or explants do not germinate, maintain viability, and are competent for transformation, for a desired length of time prior to use in this transformation method (e.g., approximately 1 hour to approximately 2 years, or approximately 24 hours to approximately 1 year, or any specific period or range of time within that broader range). The use of storable seeds or explants would enable a reliable supply of seed or explant raw materials without the need for donor plants. The ability to store mature, dry seeds relates to the natural properties of the dry, mature seeds and embryos. In other words, dry, mature seeds and / or embryo explants can also be defined in terms of quiescence, stasis, or a low metabolic state or activity. Accordingly, dried seeds or explants used in accordance with the methods of this disclosure may be defined in terms of a low metabolic state from the time of hydration and germination after the seed or embryo, and / or by a metabolic or developmental quiescence or stasis state.

[0056] According to some embodiments, hydration or germination of the explant or seed can be performed before or after the excision of the explant from the seed. In other words, apart from any pre-culture step, the seed can be absorbed or hydrated to enable the seed to begin germination and / or development before the excision of the explant, or alternatively, the dried explant is excised from the seed and then absorbed or hydrated to trigger the germination and / or development of the explant. The primed or germinated seed is then transformed without prior greening of the target tissue, which can be controlled by the amount of time and / or limited exposure to light prior to the particle impact step. However, as described above, the hydration step may instead be used solely to hydrate the dried embryo explant, making the "moistened" explant more suitable for particle impact and delivery of DNA encoding one or more genome editing reagents without embryo germination or further development (for example, the hydration or absorption step may be limited to a time such that significant developmental changes and / or germination of the embryo explant do not occur before impact).

[0057] Explants for use in embodiments of the methods provided herein may include explants from a variety of monocots and dicots, encompassing agricultural crop species (such as maize, wheat, rice, sorghum, wild oats, barley, sugarcane, guinea palm, switchgrass, cotton, canola, sugar beet, alfalfa, soybean, and other Fabaceae or legumes).

[0058] III. Genome Editing The cells, plants, plant parts, and seeds of this disclosure are produced through genome modification using site-directed integration or genome editing. Targeted modifications of the plant genome via genome editing can be used to produce crop plants with improved traits. Genome editing can be used to create one or more edits or mutations (such as altering the expression and / or activity of one or more genes, or incorporating an insertion sequence or transgene at a desired location in the plant genome) at desired target sites in the plant genome. As used herein, “site-directed integration” refers to genome editing methods and techniques that enable targeted integration or insertion of polynucleotides (e.g., insertion sequences, regulatory elements, or transgenes) into the plant genome. As provided herein, DNA molecules encoding one or more genome editing reagents can be delivered to recipient cells of an explant (such as cells related to the meristem of the explant). Guide nucleic acids and / or DNA encoding one or more genome editing reagents can be delivered to recipient cells of an explant via a transformation method without incorporating or uprooting polynucleotides or transgenes into the genome of the recipient cells. The DNA molecules encoding one or more genome editing reagents may further include (i) a transcriptable DNA sequence encoding a guide nucleic acid for a guide nuclease, (ii) a marker gene or transgene (such as a selectable or screenable marker gene), and / or (iii) a donor template.

[0059] According to many embodiments, a recombinant DNA construct or molecule is provided comprising a sequence encoding one or more genome editing reagents, the sequence being operably ligated to a promoter. The promoter may be heterogeneous with respect to the sequence encoding a site-specific nuclease. The promoter may be a plant-expressable promoter (such as a constitutive promoter, a tissue-specific or tissue-preferential promoter, a developmental stage promoter, or an inducible promoter). According to some embodiments, the recombinant DNA construct or molecule may further comprise (i) a second sequence, or a transcriptable DNA sequence encoding a guide nucleic acid (the second sequence being operably ligated to a second promoter), and / or (ii) a marker gene or transgene (which may be a selectable or screenable marker gene). The second promoter may be heterogeneous with respect to the sequence encoding the guide nucleic acid. The second promoter may be a plant-expressable promoter (such as a constitutive promoter, a tissue-specific or tissue-preferential promoter, a developmental stage promoter, or an inducible promoter). The marker gene or transgene may comprise a coding sequence operably ligated to a promoter (such as a heterogeneous promoter and / or a plant-expressable promoter). According to some embodiments, the recombinant DNA construct or molecule may further include a donor template region. The recombinant DNA constructs of this disclosure can be delivered to recipient explant cells via any transformation method known in the art (such as particle impact or bacterial-mediated transformation).

[0060] According to many embodiments, a recombinant DNA construct or molecule comprising a sequence encoding one or more genome editing reagents (the sequence being operably ligated to a promoter) is applied to or coated onto particles or beads for microparticle gun delivery, or incorporated into an explant or a transformation vector for transformation into cells or tissues of an explant. The promoter may be heterogeneous with respect to the sequence encoding the genome editing reagent. The promoter may be a plant-expressible promoter (such as a constitutive promoter, a tissue-specific promoter or a tissue-preferential promoter, a developmental stage promoter, or an inducible promoter). According to some embodiments, the recombinant DNA construct or molecule may further comprise a second sequence, or a transcriptable DNA sequence encoding a guide nucleic acid (the second sequence being operably ligated to a second promoter). The second promoter may be heterogeneous with respect to the sequence encoding the guide nucleic acid. The second promoter may be a plant-expressible promoter (such as a constitutive promoter, a tissue-specific promoter or a tissue-preferential promoter, a developmental stage promoter, or an inducible promoter). According to some embodiments, a recombinant DNA construct or molecule comprising one or more sequences encoding a genome editing reagent (the sequences being operably linked to a promoter), as well as one or more of a guide nucleic acid, a marker gene, and / or a donor DNA template, is applied to or coated onto particles or beads for microparticle gun delivery to an explant or cells or tissues of the explant.

[0061] DNA constructs or molecules encoding any suitable genome editing reagent (such as zinc finger nucleases (ZFNs), guide nucleases, TALE endonucleases (TALENs), meganucleases, recombinases, transposases, or any combination thereof) can be delivered to cells of an explant according to the methods provided herein to induce genome editing or site-directed integration at target sites in the genome of the explant cells and / or their progeny cells. In the case of guide nucleases (such as clustered regularly dispersed short palindromic repeat (CRISPR) enzymes), the DNA construct or molecule can be co-delivered with a guide nucleic acid to direct the guide nuclease to the target site. Examples of guide nucleases may also include homologs or modified versions of any known guide nuclease that share conserved amino acids and have higher percentage identity with respect to their respective protein sequences (e.g., at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity in their protein sequences across their alignment lengths).

[0062] According to some embodiments, a DNA construct or molecule encoding a genome editing reagent may be co-delivered with a donor template molecule to serve as a template for producing desired edits, mutations, or insertions in the genome at desired target sites via repair of double-strand breaks (DSBs) or nicks generated by the genome editing reagent. According to some embodiments, a DNA construct or molecule encoding a genome editing reagent may be co-delivered with a DNA molecule containing a selectable or screenable marker gene. In each case, in addition to the DNA construct or molecule encoding the genome editing reagent, one or more of the guide nucleic acid, donor template molecule, and / or DNA molecules encoding a selectable or screenable marker may be applied to or coated onto particles used for microparticle gun or particle delivery to recipient cells of the explant. According to some embodiments, a DNA construct or molecule encoding a genome editing reagent can be applied to or coated onto particles used for particle gun or particle delivery, or incorporated into a vector for transformation into one or more recipient cells of an explant, the one or more recipient cells of the explant comprising one or more DNA molecules and / or transgenes that can be stably transformed into the genome of the recipient cells prior to particle impact or transformation, wherein such DNA molecules and / or transgenes comprise or encode one or more of the following: (i) a donor template molecule provided as a template for making desired edits, mutations or insertions in the genome at desired target sites; (ii) a selectable or screenable marker gene; and / or (iii) a guide nucleic acid that directs a guide nuclease to desired target sites.

[0063] Genome editing reagents can be guide nucleases, which can function as ribonucleoprotein (RNP) complexes with guide RNA. According to some embodiments, guide nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, and Cmr. The group can be selected from 1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, CasZ, and their homologs or modified versions, and Argonaut (non-limiting examples of Argonaut proteins include Thermus thermophilus Argonaut (TtAgo), Pyrococcus furiosus Argonaut (PfAgo), Natronobacterium gregoryi Argonaut (NgAgo), and their homologs or modified versions). According to some embodiments, the guide nuclease is the Cas9 enzyme or the Cpf1 enzyme. A DNA construct or molecule encoding a guide nuclease can be delivered with or without the guide nucleic acid.

[0064] For guide nucleases, guide nucleic acid molecules may be further provided to direct the guide nuclease to a target site in the plant genome via base pairing or hybridization, thereby inducing a double-segment break (DSB) or nick at or near the target site. Guide nucleic acids may be transformed or introduced into plant cells or tissues as guide nucleic acid molecules, or as recombinant DNA molecules, constructs, or vectors containing a transcriptable DNA sequence encoding a guide nucleic acid operably linked to a promoter or a promoter expressible in plants. Promoters may be constitutive promoters, tissue-specific promoters or tissue-preferential promoters, developmental stage promoters, or inducible promoters.

[0065] As used herein, the term “guide nucleic acid” refers to a nucleic acid comprising a first segment containing a nucleotide sequence complementary to the sequence in the target nucleic acid, and a second segment that interacts with a guide nuclease protein. In some embodiments, the first segment of the guide containing the nucleotide sequence complementary to the sequence in the target nucleic acid corresponds to CRISPR RNA (crRNA repeat or crRNA repeat). In some embodiments, the second segment of the guide containing the nucleic acid sequence that interacts with the guide nuclease protein corresponds to trans-acting CRISPR RNA (tracrRNA). In some embodiments, the guide nucleic acid comprises two separate nucleic acid molecules that hybridize with each other (a polynucleotide complementary to the sequence in the target nucleic acid, and a polynucleotide that interacts with the guide nuclease protein) and is referred herein to as a “dual guide” or “two-molecule guide.” In some embodiments, the dual guide may comprise DNA, RNA, or a combination of DNA and RNA. In other embodiments, the guide nucleic acid is a single polynucleotide and is referred herein to as a “single-molecule guide” or “single guide.” In some embodiments, a single guide may include DNA, RNA, or a combination of DNA and RNA. The term “guide nucleic acid” is inclusive and refers to both bimolecule guides and single-molecule guides.

[0066] The protospacer adjacency motif (PAM) may be located in the genome directly adjacent to the 5' end of a genomic target site sequence complementary to the targeting sequence of the guide RNA, and upstream thereof (directly downstream (3') of the sense (+) strand of the genomic target site (compared to the targeting sequence of the guide RNA), as is well known in the art. See, for example, Wu, X. et al., “Target specificity of the CRISPR-Cas9 system,” Quant Biol. 2(2):59-70 (2014). The genomic PAM sequence on the sense (+) strand adjacent to the target site (compared to the targeting sequence of the guide RNA) may include 5'-NGG-3'. However, generally speaking, the corresponding sequence of the guide nucleic acid (directly downstream (3') of the targeting sequence of the guide RNA) does not have to be complementary to the genomic PAM sequence.

[0067] Guide nucleic acids are typically non-coding RNA molecules that do not code for proteins. The target sequence of the guide nucleic acid may be at least 10 nucleotides in length (e.g., 12–40 nucleotides, 12–30 nucleotides, 12–20 nucleotides, 12–35 nucleotides, 12–30 nucleotides, 15–30 nucleotides, 17–30 nucleotides, or 17–25 nucleotides in length, or approximately 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides in length). The targeter sequence may be at least 95%, at least 96%, at least 97%, at least 99%, or 100% identical or complementary to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or more consecutive nucleotides of the DNA sequence at the target site of the genome.

[0068] In addition to the targeter sequence, the guide nucleic acid may further include one or more other structural sequences or scaffold sequences that can bind to or interact with the RNA guide endonuclease. Such scaffold sequences or structural sequences may further interact with other RNA molecules (e.g., tracrRNA). Methods and techniques for designing targeting constructs and guide nucleic acids for genome editing and site-directed integration at target sites in the plant genome using guide nucleases are known in the art.

[0069] Multiple site-specific nucleases (such as recombinases, zinc finger nucleases (ZFNs), meganucleases, and TALENs) are not guided by nucleic acids; instead, they rely on their protein structure to determine their target site to induce DSBs or nicks, or they are fused, ligated, or attached to DNA-binding protein domains or motifs. The protein structure of a site-specific nuclease (or fused / attached or ligated DNA-binding domain) can target the site-specific nuclease to its target site. According to many of these embodiments, site-specific nucleases not guided by nucleic acids (such as recombinases and zinc finger nucleases (ZFNs), meganucleases, and TALENs) are designed, manipulated, and constructed according to known methods to target and bind to target sites at or near genomic loci of endogenous plant genes, thereby generating DSBs or nicks at such genomic loci and knocking out or knocking down gene expression via DSB or nick repair (which may lead to the generation of sequence mutations or insertions at the DSB or nick site via a cellular repair mechanism that can be guided by a donor template molecule).

[0070] In some embodiments, the site-specific nuclease is a recombinase. The recombinase may be a serine recombinase attached to a DNA recognition motif, a tyrosine recombinase attached to a DNA recognition motif, or other recombinase enzymes known in the art. The recombinase or transposase may be a DNA transposase or recombinase attached to or fused to a DNA-binding domain. Non-limiting examples of recombinases include tyrosine recombinases attached to DNA recognition motifs provided herein, selected from the group consisting of Cre recombinase, Gin recombinase, Flp recombinase, and Tnp1 recombinase. In one embodiment, the Cre recombinase or Gin recombinase provided herein is ligated to a zinc finger DNA-binding domain, a TALE DNA-binding domain, or a Cas9 nuclease. In another embodiment, the serine recombinase attached to the DNA recognition motif provided herein is selected from the group consisting of PhiC31 integrase, R4 integrase, and TP-901 integrase. In another embodiment, the DNA transposase attached to the DNA binding domain provided herein is selected from the group consisting of TALE-piggyBac and TALE mutagenesis.

[0071] Site-specific nucleases can be zinc finger nucleases (ZFNs). ZFNs are synthetic proteins consisting of an engineered zinc finger DNA-binding domain fused to a cleavage domain (or half of a cleavage domain) that may be derived from a restriction enzyme (e.g., FokI). The DNA-binding domain can be canonical (C2H2) or non-canonical (e.g., C3H or C4). The DNA-binding domain may contain one or more zinc fingers (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more) that are dependent on the target site. Multiple zinc fingers in the DNA-binding domain can be separated by a linker sequence. ZFNs can be designed to cleave almost any stretch of double-stranded DNA by modification of the zinc finger DNA-binding domain. ZFNs dimerize from monomers consisting of non-specific DNA cleavage domains (e.g., derived from the FokI nuclease) fused to a DNA-binding domain containing a zinc finger array engineered to bind to a target site DNA sequence. The DNA-binding domain of a ZFN can typically consist of 3-4 (or more) zinc fingers. Compared to the starting point of the zinc finger α-helix, the amino acids at positions -1, +2, +3, and +6 (which contribute to site-specific binding to the target site) can be altered and customized to fit a specific target sequence. Other amino acids can form a consensus skeleton to generate ZFNs with different sequence specificities.

[0072] Methods and rules for designing ZFNs to target and bind to specific target sequences are known in the art. See, for example, U.S. Patent Applications 2005 / 0064474, 2009 / 0117617, and 2012 / 0142062. The FokI nuclease domain requires dimerization to cleave DNA, and therefore two ZFNs with C-terminal regions are needed to bind the opposite DNA strands of the cleavage site (separated at 5-7 bp). If the two ZF binding sites are palindromic, the ZFN monomer can cleave the target site. When used herein, ZFN broadly encompasses monomeric ZFNs that can cleave double-stranded DNA without assistance from another ZFN. The term ZFN may also be used to refer to one or both members of a pair of ZFNs that have been engineered to work together to cleave DNA at the same site. While not limited by any particular theory, the DNA binding specificity of zinc finger domains can be remanufactured using one of several methods, so that customized ZFNs can theoretically be constructed to target almost any target sequence (e.g., genes in or near a plant genome). Publicly available methods for manipulating zinc finger domains include Context-dependent Assembly (CoDA), Oligomerized Pool Engineering (OPEN), and Modular Assembly. In one embodiment, the methods and / or compositions provided herein comprise one or more, two or more, three or more, four or more, or five or more ZFNs. In another embodiment, the ZFNs provided herein are capable of generating targeted DSBs or nicks.

[0073] Site-specific nucleases can be TALEN enzymes. TALENs are artificial restriction enzymes produced by fusing a transcription activator-like effector (TALE) DNA-binding domain to a nuclease domain (e.g., FokI). When each member of the TALEN pair binds to a DNA site adjacent to the target site, the FokI monomer dimerizes, causing a cleavage of double-stranded DNA at the target site. In addition to the wild-type FokI cleavage domain, variants of the FokI cleavage domain with mutations have been designed to improve cleavage specificity and activity. The FokI domain functions as a dimer, requiring two constructs with unique DNA-binding domains for the target site in the genome, oriented and spaced appropriately. Both the number of amino acid residues between the TALEN DNA-binding domain and the FokI cleavage domain, and the number of bases between the two separate TALEN binding sites, are parameters for achieving high levels of activity.

[0074] TALENs are artificial restriction enzymes produced by fusing a transcription activator-like effector (TALE) DNA-binding domain to a nuclease domain. In some embodiments, the nuclease is selected from the group consisting of PvuII, MutH, TevI, FokI, AlwI, MlyI, SbfI, SdaI, StsI, CleDORF, Clo051, and Pept071. When each member of a TALEN pair binds to a DNA site adjacent to the target site, the FokI monomer dimerizes, causing a break in double-stranded DNA at the target site. As used herein, the term TALEN broadly encompasses monomeric TALENs that can break double-stranded DNA without the assistance of another TALEN. The term TALEN also refers to one or both members of a pair of TALENs that work together to break DNA at the same site.

[0075] Transcription activator-like effectors (TALEs) can be manipulated to bind to virtually any DNA sequence (e.g., at or near genomic loci of genes in plants). TALEs have a central DNA-binding domain composed of 13–28 repeat monomers of 33–34 amino acids. The amino acids in each monomer are highly conserved, except for the hypervariable amino acid residues at positions 12 and 13. Two variable amino acids are called repeat variable duos (RVDs). The amino acid pairs NI, NG, HD, and NN of the RVDs preferentially recognize adenine, thymine, cytosine, and guanine / adenine, respectively, and by modulating the RVDs, a sequence of DNA bases can be recognized. This simple relationship between amino acid sequences and DNA recognition has made it possible to manipulate specific DNA-binding domains by selecting combinations of repeat segments containing appropriate RVDs.

[0076] Beyond the wild-type FokI cleavage domain, variants of the FokI cleavage domain with mutations have been designed to improve cleavage specificity and activity. The FokI domain functions as a dimer, requiring two constructs with unique DNA-binding domains for a site in the target genome, oriented and spaced appropriately. Both the number of amino acid residues between the TALEN DNA-binding domain and the FokI cleavage domain, and the number of bases between the two separate TALEN-binding sites, are parameters for achieving high levels of activity. The PvuII, MutH, and TevI cleavage domains are useful alternatives to FokI and its variants for use with TALE. PvuII functions as a highly specific cleavage domain when coupled to TALE (see Yank et al. 2013. PLoS One. 8:e82539). MutH can introduce specific nicks into strands in DNA (see Gabsalilow et al. 2013. Nucleic Acids Research. 41:e83). TevI introduces double-strand breaks in DNA at targeted sites (see Beurdeley et al., 2013. Nature Communications. 4:1762).

[0077] The relationship between the amino acid sequence of the TALE-binding domain and DNA recognition enables designable proteins. TALE constructs can be designed using software programs such as DNAWorks. Other methods for designing TALE constructs are known to those skilled in the art. See Doyle et al., Nucleic Acids Research (2012) 40:W117-122; Cermak et al., Nucleic Acids Research (2011) 39:e82; and tale-nt.cac.cornell.edu / about. In another embodiment, the TALENs provided herein are capable of generating targeted DSBs.

[0078] Site-specific nucleases can be meganucleases. Meganucleases commonly identified in microorganisms (such as the LAGLIDADG family of homing endonucleases) are unique enzymes that possess high activity and long recognition sequences (>14 bp), resulting in site-specific digestion of target DNA. Engineered versions of naturally occurring meganucleases typically have extended DNA recognition sequences (e.g., 14–40 bp). According to some embodiments, meganucleases may comprise scaffold or base enzymes selected from the group consisting of I-CreI, I-CeuI, I-MsoI, I-SceI, I-AniI, and I-DmoI. Because the DNA recognition and cleavage functions of meganucleases are intertwined within a single domain, manipulating meganucleases can be more challenging than manipulating ZFNs and TALENs. Specialized methods of mutagenesis and high-throughput screening have been used to generate novel meganuclease variants that recognize unique sequences and retain improved nuclease activity. Therefore, meganucleases can be selected or manipulated to bind to genomic target sequences in plants (e.g., at or near genomic loci of genes). In another embodiment, the meganucleases provided herein are capable of generating targeted DSBs.

[0079] According to some embodiments, a donor template may be co-delivered to the recipient cells of the explant along with a DNA construct or molecule encoding a genome editing reagent to serve as a template for generating desired edits during the repair of double-strand breaks (DSBs) or nicks at target sites in the recipient cell genome by the genome editing reagent. Alternatively, the donor template may already be present in the recipient cells of the explant. Similarly, for guide nucleases, a transcriptable DNA sequence or transgene encoding the guide nucleic acid may also be co-delivered to the recipient cells of the explant along with a DNA construct or molecule encoding the guide nuclease to serve as a guide for directing the guide nuclease to produce double-strand breaks (DSBs) or nicks at desired loci or target sites in the recipient cell genome. Alternatively, the guide nucleic acid, and / or the DNA molecule or transgene containing the transcriptable DNA sequence encoding the guide nucleic acid, may already be present and / or expressed by the recipient cells of the explant.

[0080] According to some embodiments, (i) a DNA construct or molecule encoding a genome editing reagent, guide nucleic acid, and donor template may be applied to or coated on particles for microparticle gun delivery to recipient cells, or (ii) a DNA construct or molecule encoding a genome editing reagent and / or guide RNA may be applied to or coated on particles for microparticle gun delivery to recipient cells, and the donor template may be optionally present or expressed in recipient cells, or (iii) a DNA construct or molecule encoding a genome editing reagent and / or donor template may be applied to or coated on particles for microparticle gun delivery to recipient cells. (i) the guide nucleic acid may be coated onto the recipient cell, the guide nucleic acid may be optionally present in or expressed in the recipient cell, or (iv) the guide nucleic acid and / or donor template may be applied to or coated onto particles for microparticle gun delivery to the recipient cell, the genome editing reagent, or the DNA construct or molecule encoding the genome editing reagent may be present in or expressed in the recipient cell, in each case of (i), (ii), (iii), or (iv), the genome editing reagent is used to create a double-strand break (DSB) or nick at a desired locus or target site in the recipient cell genome to produce template-mediated or template-independent editing or mutation at a desired location in the genome of the recipient plant cell.

[0081] Any site or locus within the plant genome may be selected as a potential site for genome editing (or gene editing) or site-directed integration of a transgene, construct, or transcriptional DNA sequence. For genome editing and site-directed integration, a double-strand break (DSB) or nick may be initially produced at the selected genomic locus by a genome editing reagent (e.g., zinc finger nuclease (ZFN), engineered or innate meganuclease, TALE endonuclease, or guide endonuclease (e.g., Cas9 or Cpf1)). Any method known in the art for site-directed integration may be used. In the presence of a donor template molecule containing the insertion sequence, the DSB or nick may be repaired by homologous recombination between the donor template and homologous arms of the plant genome, or by non-homologous end joining (NHEJ), resulting in site-directed integration of the insertion sequence into the plant genome and generating a targeted insertion event at the DSB or nick site. Therefore, site-specific insertion or integration of the transgene, transcriptional DNA sequence, construct, or sequence can be achieved if the transgene, transcriptional DNA sequence, construct, or sequence is located within the insertion sequence of the donor template.

[0082] Targeted mutations can also be introduced into the plant genome using the introduction of DSBs or nicks. According to this approach, mutations (deletions, insertions, inversions, and / or substitutions, etc.) can be introduced at a target site via incomplete repair of DSBs or nicks to produce gene knockout or knockdown. Such mutations may, in some cases, be generated by incomplete repair of the targeted locus without the use of a donor template molecule. Gene "knockout" can be achieved by inducing a DSB or nick at or near the gene's endogenous locus, resulting in non-expression of the protein or expression of a non-functional protein; gene "knockdown" can be achieved in a similar manner by inducing a DSB or nick at or near the gene's endogenous locus, which is incompletely repaired at a site that does not affect the gene's coding sequence, resulting in loss of function of the encoded protein. For example, the site of a DSB or nick within an endogenous locus may be located upstream or in the 5' region of the gene (e.g., promoter and / or enhancer sequences) and may affect or reduce the level of expression. Similarly, such targeted knockout or knockdown mutations of a gene can be generated by a donor template molecule that directs a specific or desired mutation at or near the target site via DSB or nick repair. The donor template molecule may contain a homologous sequence that, with or without insertions, contains one or more mutations (one or more deletions, insertions, inversions, and / or substitutions, etc.) at or near the DSB or nick site compared to the targeted genomic sequence. For example, a targeted knockout mutation of a gene can be achieved by substituting, inserting, deleting, or inverting at least a portion of the gene (for example, by introducing a frameshift or immature stop codon into the gene's coding sequence). A partial deletion of a gene can also be introduced by generating a DSB or nick at two target sites, causing a deletion in an intervening target region adjacent to the target sites.

[0083] As used herein, a “donor molecule,” “donor template,” or “donor template molecule” (collectively, “donor template”), which may be a recombinant polynucleotide, DNA, or RNA donor template or sequence, is defined as a nucleic acid molecule having a homologous nucleic acid template or sequence (e.g., homologous sequence) and / or an insertion sequence for site-directed, targeted insertion or recombination into the genome of a plant cell via repair of nicks or double-strand DNA breaks in the genome of the plant cell. A donor template may be an isolated DNA molecule containing one or more homologous sequences and / or an insertion sequence for targeted integration, or a donor template may be a sequence portion (donor template region) of a DNA molecule further containing one or more other expression cassettes, genes / transgenes, and / or transcriptional DNA sequences. For example, a “donor template” may be used as a template for site-directed integration of a transgene or repressor construct, or for introducing mutations (insertions, deletions, substitutions, etc.) into a target site in the genome of a plant. The targeted genome editing techniques provided herein may involve the use of one or more, two or more, three or more, four or more, or five or more donor molecules or templates. A “donor template” may be a single-stranded or double-stranded DNA or RNA molecule or plasmid. The “insertion sequence” of the donor template is a sequence designed for targeted insertion into the genome of a plant cell, and may be of any preferred length.For example, the insertion sequences for the donor mold are between 2 and 50,000, between 2 and 10,000, between 2 and 5,000, between 2 and 1,000, between 2 and 500, between 2 and 250, between 2 and 100, between 2 and 50, between 2 and 30, between 15 and 50, between 15 and 100, between 15 and 500, between 15 and 1,000, between 15 and 5,000, between 18 and 30, between 18 and 26, between 20 and 26, between 20 and 50, between 20 and 100, between 20 and 250, between 20 and 500, and between 20 and 500. The length of a nucleotide or base pair can be between 1,000, between 20 and 5,000, between 20 and 10,000, between 50 and 250, between 50 and 500, between 50 and 1,000, between 50 and 5,000, between 50 and 10,000, between 100 and 250, between 100 and 500, between 100 and 1,000, between 100 and 5,000, between 100 and 10,000, between 250 and 500, between 250 and 1,000, between 250 and 5,000, or between 250 and 10,000. The donor template also has at least one homologous sequence or homologous arm(s) such that homologous recombination directs the insertion of a mutation or insertion sequence into a target site in the plant genome, and the homologous sequence or homologous arm(s) are identical or complementary, or have percent identity or percent complementarity to a sequence at or near the target site in the plant genome. If the donor template contains homologous arm(s) and an insertion sequence, the homologous arm(s) will be adjacent to or surround the insertion sequence in the donor template.

[0084] According to some embodiments, the donor template comprises a “donor template region” of a recombinant polynucleotide molecule or construct that functions as a donor template for site-specific integration or template-mediated repair of an insertion sequence, and the recombinant polynucleotide molecule or construct further comprises other elements outside the donor template region that may be independent of the donor template. For example, the recombinant polynucleotide molecule or construct may comprise the “donor template region” as well as a transcriptable DNA sequence encoding one or more transgenes (such as selectable markers) and / or non-coding RNA molecules (such as guide RNA or RNA molecules for repression of a target gene).

[0085] The donor template insertion sequence may contain one or more genes or sequences encoding a transcribed non-coding RNA or mRNA sequence and / or a translated protein sequence, respectively. The transcribed sequence or gene of the donor template may encode a protein or a non-coding RNA molecule. The non-coding RNA molecule may be, for example, a guide RNA or an RNA molecule that targets a gene for repression (e.g., microRNA (miRNA), small interfering RNA (siRNA), antisense RNA strand, reverse repeat, etc.). The donor template insertion sequence may contain a polynucleotide sequence that does not contain a functional gene or the entire gene sequence (e.g., the donor template may simply contain a regulatory sequence such as a promoter sequence, or only a portion of a gene or coding sequence), or it may not contain any identifiable gene expression element or any actively transcribed gene sequence. Furthermore, the donor template may be linear or circular, or single-stranded or double-stranded. The donor template may be delivered to the cell as a DNA or RNA molecule expressed from the transgene. The donor template may be delivered to cells as a naked nucleic acid molecule or as a complex with one or more delivery agents (e.g., liposomes, proteins, poloxamers, protein-encapsulated T chains). The insertion sequences of the donor templates provided herein include a transcribed DNA sequence that can be transcribed into an RNA molecule (which may be non-coding or protein-coding), and the transcribed DNA sequence may be operably ligated to a promoter and / or other regulatory sequence (such as a constitutive, inducible, or tissue-specific promoter).

[0086] According to some embodiments, the donor template does not have to include an insertion sequence, but instead may include one or more homologous sequences that contain one or more mutations (insertions, deletions, substitutions, etc.) compared to the genomic sequence at a target site in the plant genome (e.g., in or near a gene in the plant genome). Alternatively, the donor template may include an insertion sequence that does not contain a coding sequence or a transcriptable DNA sequence, and the insertion sequence can be used to introduce one or more mutations into a target site in the plant genome (e.g., in or near a gene in the plant genome).

[0087] The donor templates provided herein may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 genes(s) or transgenes(s) and / or transcriptional DNA sequences(s). Alternatively, the donor template may not comprise genes, transgenes, or transcriptional DNA sequences. Examples of genes / transgenes or transcriptional DNA sequences of the donor template may include, but are not limited to, insecticide resistance genes, herbicide resistance genes, nitrogen use efficiency genes, water use efficiency genes, yield-enhancing genes, nutrient-value genes, DNA-binding genes, selectable marker genes, RNAi or repression constructs, genome editing reagents, single guide RNAs for CRISPR / Cas9 systems, geminivirus-based expression cassettes, or plant virus expression vector systems. According to other embodiments, the insertion sequences of the donor template may comprise protein-coding sequences or transcriptional DNA sequences encoding non-coding RNA molecules that can target endogenous genes for repression. A donor template may include a promoter (such as a constitutive promoter, tissue-specific or tissue-preferential promoter, developmental stage promoter, or inducible promoter) operably linked to a coding sequence, gene, or transcriptable DNA sequence. A donor template may include a leader, enhancer, promoter, transcription start site, 5'-UTR, one or more exons, one or more introns, transcription termination sites, regions, or sequences, 3'-UTR, and / or polyadenylation signals (each of which may be operably linked to a coding sequence), a gene (or transgene), or a transcriptable DNA sequence encoding non-coding RNA, guide nucleic acid, mRNA, and / or protein.

[0088] According to this embodiment, a portion of a recombinant donor template polynucleotide molecule (insertion sequence) may be inserted or incorporated at a desired site or locus within the plant genome via genome editing. The donor template insertion sequence may include a transgene or construct (such as a protein-coding transgene or a transcriptable DNA sequence encoding a non-coding RNA molecule that targets an endogenous gene for repression). The donor template may also have one or two homologous arms adjacent to the insertion sequence to enhance targeted insertion events via homologous recombination and / or homologous directional repair. Each homologous arm is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 99%, or 100% identical or complementary to at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 500, at least 1000, at least 2500, or at least 5000 consecutive nucleotides of a target DNA sequence in the genome of a plant cell. According to some embodiments, recombinant DNA donor template molecules for site-directed or targeted integration of insertion sequences and / or recombination of homologous sequences(s) into a plant genome may be co-delivered with a DNA construct or molecule encoding a genome editing reagent, the insertion sequence may include a transgene or construct (such as a transgene or a transcriptable DNA sequence encoding a non-coding RNA molecule that targets an endogenous gene for repression). The recombinant DNA donor template may also include a transgene encoding a selectable or screenable marker gene and / or guide nucleic acid, the marker gene and the transgene encoding the guide nucleic acid, each of which may be operably linked to a promoter and / or other expression regulatory element expressible in the plant.

[0089] When used herein, “target site” for genome editing or site-directed integration refers to the location of a polynucleotide sequence in a plant genome that is bound and cleaved by a genome editing reagent to introduce a double-strand break, single-strand nick, or other modification (such as deamination) into the nucleic acid backbone of the polynucleotide sequence and / or its complementary DNA strand within the plant genome. A target site may contain at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 29, or at least 30 consecutive nucleotides. For guide nucleases, a “target site” may contain a sequence of a complementary strand of either a double-stranded nucleic acid (DNA) molecule or a chromosome at the target site. Site-specific nucleases may bind to target sites via non-coding guide nucleic acids (e.g., CRISPR RNA (crRNA) or single guide RNA (sgRNA) as further described herein, without limitation). The targeter sequences of the guide nucleic acids provided herein may be complementary to the target site (e.g., complementary to a double-stranded nucleic acid molecule or any strand of a chromosome at the target site). It will be recognized that perfect identity or complementarity of the targeter sequence of the guide nucleic acid is not required for binding to or hybridizing with the target site. For example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 mismatches (or more) may be acceptable between the target site and the targeter sequence of the guide nucleic acid. The term "target site" also refers to the location of a polynucleotide sequence within a plant genome that is bound and cleaved by any other genome editing reagent (such as meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), etc.), which does not necessarily require guidance from a guide nucleic acid molecule, thereby introducing double-strand breaks, single-strand nicks, or other modifications into the polynucleotide sequence and / or its complementary DNA strand.

[0090] As used herein, “target region” or “targeted region” refers to a sequence or region of polynucleotides in which two or more target sites are adjacent. In some embodiments, though not limited to those embodiments, a target region may undergo mutation, base modification, deletion, insertion, or inversion, following repair of double-strand breaks or nicks at two target sites. As used herein, “adjacent” refers to two or more target sites of a polynucleotide sequence or molecule surrounding a target region, along with one target site on each side of the target region.

[0091] A method for producing transgenic or genome-edited plants, plant parts, and seeds by delivery of a DNA construct or molecule encoding a genome editing reagent into at least one cell of a mature and / or dried excised explant is provided herein, along with various culture and processing steps described herein for developing or regenerating genome-edited or transgenic plants. Transgenic or genome-edited plants, plant parts, and seeds produced according to this method are further provided. According to one aspect of this disclosure, plants or their offspring plants developed or regenerated from explants subjected to particle impact or transformation by a DNA construct or molecule encoding a genome editing reagent can be screened or selected based on markers, traits, or phenotypes produced in the developed or regenerated plants or their offspring plants, plant parts, or seeds by editing or mutation, or by site-directed integration such as insertion sequences or transgenes. If a given mutation, edit, trait, or phenotype is recessive, then one or more generations or crosses (e.g., autopolation) from the initial R0 plant may be required to produce a homozygous plant for the edit or mutation so that the trait or phenotype can be observed. The offspring plants (plants propagated from R1 seeds or plants in subsequent generations, etc.) can be tested for conjugation using any known conjugation assay (by using SNP assays, DNA sequencing, thermal amplification or PCR, and / or Southern blotting, etc., which allow for differentiation between heterozygotes, homozygotes and wild-type plants).

[0092] In a further embodiment, one or more tissues or cells of a plant that has developed or regenerated from an explant that has undergone particle impact or transformation with DNA encoding a genome editing reagent, or of its offspring plants, or of a part or seed of the aforementioned plant, may be screened or selected based on molecular assays that detect the presence of edits or mutations, or site-directed integrations such as insertion sequences or transgenes. Assays that may be used to detect the presence of transgenes introduced by editing, mutation, or site-directed integration include, for example, molecular biological assays (Southern and Northern blotting, PCR, FLA, and DNA sequencing); and biochemical assays (e.g., detecting the presence of protein products by immunological means (ELISA and Western blotting), or by enzyme function or in vitro analysis). Alternatively, a plant that has developed or regenerated from an explant that has undergone particle impact or transformation with DNA encoding a genome editing reagent, or of its offspring plants or seeds, may be screened or selected based on phenotypes or traits that may be desired or predicted.

[0093] IV. Definition The following definitions are provided to define and clarify the meaning of these terms and to guide those skilled in the art in understanding this disclosure, with reference to relevant embodiments of this disclosure as used herein. Unless otherwise noted, terms should be understood in accordance with their conventional meanings and usages in the relevant technical fields, particularly in the fields of molecular biology and plant transformation.

[0094] An "embryo" is a part of a plant seed consisting of precursor tissue (e.g., tissue relating to the meristem) that can develop into all or part of a mature plant. An "embryo" may further encompass a part of the plant embryo.

[0095] A "meristem" or "tissue relating to a meristem" comprises undifferentiated cells or cells relating to a meristem that are capable of differentiating to produce one or more types of plant parts, tissues, or structures (such as buds, stems, roots, leaves, seeds, all or parts thereof).

[0096] As used herein, the term “genome editing reagent” refers to any enzyme capable of modifying nucleotide sequences in a sequence-specific manner. In some embodiments, the genome editing reagent modifies the genome by inducing single-strand breaks. In some embodiments, the genome editing reagent modifies the genome by inducing double-strand breaks. In some embodiments, the genome editing reagent comprises cytidine deaminase. In some embodiments, the genome editing reagent comprises adenine deaminase. In this disclosure, genome editing reagents include endonucleases, recombinases, transposases, deaminases, helicases, and any combination thereof. In some embodiments, the genome editing reagent is a sequence-specific nuclease.

[0097] In one embodiment, genome editing reagents include meganucleases, zinc finger nucleases (ZFNs), activator-like effector nucleases (TALENs), argonauts (non-limiting examples of argonaut proteins include Thermus thermophilus argonaut (TtAgo), Pyrococcus furiosus argonaut (PfAgo), and Natronobacterium gregoryi argonaut (NgAgo)), and guide nucleases such as CRISPR-related nucleases (non-limiting examples of CRISPR-related nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cpf1 (also known as Cas12a), Csy1, Csy2, Csy3, Cse1, and Cse2 It is an endonuclease selected from Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, CasX, CasY, its homologs, or modified versions thereof.

[0098] In some embodiments, the genome editing reagent includes a DNA-binding domain operably linked to a deaminase. In some embodiments, the DNA-binding domain is derived from a CRISPR-related protein. In some embodiments, the genome editing reagent includes uracil DNA glycosylase (UGI). In some embodiments, the deaminase is cytidine deaminase. In some embodiments, the deaminase is adenine deaminase. In some embodiments, the deaminase is APOPEC deaminase. In some embodiments, the deaminase is activation-inducible cytidine deaminase (AID). In some embodiments, the DNA-binding domain is a zinc finger DNA-binding domain, a TALE DNA-binding domain, a Cas9 nuclease, a Cpf1 nuclease, a catalytically inactive Cas9 nuclease, a catalytically inactive Cpf1 nuclease, a Cas9 nickasase, or a Cpf1 nickasase.

[0099] In some embodiments, the genome editing reagent is a recombinase. Non-limiting examples of recombinases include tyrosine recombinases attached to the DNA recognition motif provided herein, selected from the group consisting of Cre recombinase, Gin recombinase, Flp recombinase, and Tnp1 recombinase. In one embodiment, the Cre recombinase or Gin recombinase provided herein is ligated to a zinc finger DNA-binding domain, a TALE DNA-binding domain, or a Cas9 nuclease. In another embodiment, the serine recombinase attached to the DNA recognition motif provided herein is selected from the group consisting of PhiC31 integrase, R4 integrase, and TP-901 integrase. In yet another embodiment, the DNA transposase attached to the DNA-binding domain provided herein is selected from the group consisting of TALE-piggyBac and TALE mutagenic genes.

[0100] The terms "regeneration" and "regeneration" refer to the process of propagating or developing a plant from one or more plant cells through one or more culture steps.

[0101] The term “recombinant” refers to polynucleotide (DNA or RNA) molecules, proteins, constructs, vectors, etc., which are artificial and not normally found in nature, and / or exist in circumstances in which they are not normally found in nature, and include polynucleotide (DNA or RNA) molecules, proteins, constructs, etc., which contain together combinations of two or more polynucleotide or protein sequences that do not naturally exist in the same manner without human intervention, and polynucleotide molecules, proteins, constructs, etc., which are operably linked but contain at least two polynucleotide or protein sequences that are heterogeneous to each other. For example, the term “recombinant” may refer to any combination of two or more DNA or protein sequences in the same molecule (e.g., plasmid, construct, vector, chromosome, protein, etc.) which are artificial and not normally found in nature. When used in this definition, the phrase “not normally found in nature” means not found naturally without human introduction. Recombinant polynucleotide or protein molecules, constructs, etc., may include polynucleotide or protein sequences(or sequences) that (i) are separated from other polynucleotide or protein sequences(or sequences) that are in close proximity to each other in nature, and / or (ii) are adjacent to (or contiguous with) other polynucleotide or protein sequences(or sequences) that are not in close proximity to each other in nature. Such recombinant polynucleotide molecules, proteins, constructs, etc., may also refer to genetically engineered and / or constructed polynucleotide or protein molecules or sequences outside of cells. For example, recombinant DNA molecules may include any engineered plasmid or artificial plasmid, vector, etc., and may encompass linear or circular DNA molecules. Such plasmids, vectors, etc., may contain various maintenance elements, including not only prokaryotic origins of replication and selectable markers, but also one or more transgenes or expression cassettes, and possibly plant selectable marker genes, etc.

[0102] The term "operably linked" refers to a functional linkage between a promoter or other regulatory element and a transcriptional DNA sequence or gene coding sequence (or transgene) in which the promoter or other regulatory element acts or functions to initiate, assist, influence, cause, and / or promote the transcription and expression of the relevant transcriptional DNA sequence or coding sequence in at least specific cells, tissues, developmental stages, and / or conditions.

[0103] As is commonly understood in the art, the term “promoter” can generally refer to a DNA sequence containing an RNA polymerase binding site, a transcription initiation site, and / or a TATA box, which supports or enhances the transcription and expression of a related transcriptable polynucleotide sequence and / or gene (or transgene). Promoters can be synthetically produced from, modified from, or derived from known or naturally occurring promoter sequences or other promoter sequences. Promoters can also include chimeric promoters, which include combinations of two or more heterogeneous sequences. Promoters of this disclosure can therefore include variants of promoter sequences that are similar in composition to, but not identical to, other promoter sequences (plural) known or provided herein. Promoters can be classified according to various criteria relating to the pattern of expression of the related coding sequence or transcriptable sequence or gene (including transgenes) operably linked to the promoter, such as constitutive, developmental, tissue-specific, inducible, etc. A promoter that drives expression in all or most tissues of a plant is referred to as a “constitutive” promoter. Promoter that drives expression during a specific period or stage of development is called a “developmental” promoter. Promoter that drives enhanced expression in a specific plant tissue compared to other plant tissues is called a “tissue-promoting” promoter or “tissue-preferential” promoter. Thus, a “tissue-preferential” promoter causes relatively higher or selective expression in a specific tissue (or more) of the plant, but with lower levels of expression in other tissues (or more) of the plant. Promoter that is expressed in a specific tissue (or more) of the plant and is little to no expression in other tissues of the plant is called a “tissue-specific” promoter. An “inducible” promoter is one that initiates transcription in response to environmental stimuli (such as cold, drought, or light) or other stimuli (such as wounds or chemical application). Promoter can also be classified in terms of its origin (heterospecific, homospecific, chimeric, synthetic, etc.).

[0104] As used herein, “plant-expressible promoter” means a promoter that can initiate, assist, influence, cause, and / or promote the transcription and expression of a relevant transcriptable DNA sequence, coding sequence, or gene in a plant cell or tissue.

[0105] With respect to a polynucleotide sequence (e.g., a transgenic DNA sequence, coding sequence, or gene), the term “heterogeneous” refers to a promoter or regulatory sequence that is not naturally occurring without human introduction and is not operably ligated to such polynucleotide sequence (e.g., the promoter or regulatory sequence has a different origin than the polynucleotide sequence, and / or is not naturally present in the plant species transformed by the promoter or regulatory sequence). Similarly, with respect to a polynucleotide sequence (e.g., a transgene, coding sequence, or transgenic DNA sequence), “heterogeneous promoter” or “heterogeneous, plant-expressable promoter” means a promoter or plant-expressable promoter that is not naturally occurring without human introduction and is not adjacent to and / or operably ligated to the polynucleotide sequence.

[0106] In some embodiments, the terms “a,” “an,” and “the,” as well as similar references used in the context describing a particular embodiment (in particular in the specific context of the following claims), may be interpreted as covering both singular and plural unless otherwise specifically noted. In some embodiments, the term “or” is used herein to mean “and / or” unless it is expressly noted that it refers only to the alternatives or that the alternatives are mutually exclusive.

[0107] The terms “comprise,” “have,” and “include” are open-ended linking verbs. Any one or more forms or tenses of these verbs (such as “comprises,” “comprising,” “has,” “having,” “includes,” and “including”) are also open-ended. For example, any method of “comprises,” “has,” or “includes” one or more steps is not limited to possessing only those one or more steps, but may also cover other unlisted steps. Similarly, any composition or device of “comprises,” “has,” or “includes” one or more features is not limited to possessing only those one or more features, but may also cover other unlisted features.

[0108] When the terms “percent identity,” “% identity,” or “percent identical” are used herein with reference to two or more nucleotide or protein sequences, they are calculated by (i) comparing two optimally aligned sequences across a comparison window, (ii) determining the number of positions in which identical nucleic acid bases (for nucleotide sequences) or amino acid residues (for proteins) occur in both sequences to obtain the number of matching positions, (iii) dividing the number of matching positions by the sum of the number of positions in the comparison window, and (iv) multiplying this quotient by 100% to obtain the percentage identity. If “percent identity” is calculated with respect to a reference sequence without specifying a particular comparison window, then the percentage identity is determined by dividing the number of matching positions across the alignment region by the sum of the lengths of the reference sequences. For example, the “comparison window” may be defined as the alignment region, in which case “percent identity” is also referred to as “alignment percent identity.” Therefore, as used herein, when the two sequences (query and subject) are optimally aligned (with tolerances for gaps in their alignment), the "percent identity" of the query sequence is equal to the number of identical positions between the two sequences divided by the sum of the number of positions in the query sequence over its length (or comparison window), and then multiplied by 100%.

[0109] According to some embodiments, particle complexes or compositions and formulations may contain, possibly with other components, a DNA construct or molecule encoding an "effective amount" or "effective concentration" of site-specific nuclease for editing a plant genome. The effective amount or concentration of the particle composition or formulation depends on a number of factors, such as the type, size, and quantity of particles to which the pre-assembled nuclease composition or formulation is applied, the desired genome editing efficiency, the identity and quantity of other components in the composition or formulation, the specific plant species, the type of plant material used (e.g., dry excised explants, wet excised embryos, etc.), and the specific conditions under which the composition or formulation is applied to the plant material (e.g., temperature, culture, etc.).

[0110] The compositions in some embodiments may further include an agriculturally acceptable carrier or material in combination with the particle composition. When used herein, the term “agriculturally acceptable” with respect to a carrier or material means, as in the case, that (i) it is compatible with the other components of the particle / nuclease composition for at least the purpose for which the particle / nuclease composition is used, (ii) it can be included in the particle composition to effectively and practically deliver the particle composition to plant material (e.g., dried excised explants), and (iii) it is not harmful to the plant material to which the composition is applied (at least in the manner and amount applied to or associated with the plant material).

[0111] All methods described herein may be performed in any preferred order unless otherwise indicated herein or unless it is clearly inconsistent with the context. The use of any and all embodiments, or any exemplary language provided herein with respect to a particular embodiment (e.g., “etc.”), is solely intended to illustrate the disclosure and does not imply any limitation to the scope of the disclosure unless otherwise stated in the claims.

[0112] While this disclosure has been described in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the spirit and scope of this disclosure as further defined in the appended claims. Furthermore, it should be recognized that all embodiments in this disclosure, including the following, are provided as non-limiting embodiments. [Examples]

[0113] Example 1. Preparation of beads and carrier sheets for impacting soybean explants. The following is an example of a protocol for preparing beads and carrier sheets for impacting explants. Prepare particles or beads and carrier sheets for impacting dried embryo explants from soybean seeds using a PDS1000 helium particle gun according to the following protocol. Weigh 50 mg of gold or tungsten particles into a clean DNase / RNase-free tube. Wash with 1 ml of 100% ethanol by sonication, then pelletize the particles by short-term centrifugation to remove the ethanol. Resuspend the particles in 1 ml of 100% ethanol and store at -20°C for later use. Resuspend the particles by sonication before use. Transfer 42 μl of gold or tungsten particles to a new tube, pelletize by centrifugation to remove the ethanol. Add 500 μl of sterile water and resuspend the particles by sonication. Pellet the particles by centrifugation to remove the water. Add 25 μl of water to the tube, wash the particles with a pipette tip, and then resuspend them by sonication.

[0114] In soybeans, there are two PDS genes, GmPDS11 (Glyma.11G253000, SEQ ID NO: 1) and GmPDS18 (Glyma.18G003900, SEQ ID NO: 2), located on chromosome 11 (chr11) and chromosome 18 (chr18), respectively. Three recombinant DNA constructs were designed to target the conserved regions of GmPDS11 and GmPDS18, as shown in Figure 1A, each containing a transcribed DNA sequence encoding one of three guide RNAs (gRNA1, gRNA2, or gRNA3 (SEQ ID NOs: 3-5)). Recombinant DNA molecules or fragments were constructed, each containing an expression cassette or transgene encoding Cas9 under a strong constitutive promoter and a transcribed DNA sequence encoding one of the three gRNAs under the soybean U6 promoter, as shown in Figure 1B. As further shown in Figure 1B, these recombinant DNA molecules also contained expression cassettes or transgenes for GUS and aadA. Recombinant DNA molecules for Cas9, gRNA, GUS, and aadA expression were released as linear fragments from the recombinant DNA construct-containing DNA vector using NotI restriction enzymes and I-CeuI restriction enzymes. The presence of left-hand (LB) and right-hand (RB) boundaries is indicated by the excision of these DNA molecules or fragments from the T-DNA vector, but this should not affect these experiments as the fragments are delivered to explant cells via particle impact.

[0115] Add recombinant DNA molecules, constructs, or fragments to a tube, possibly together with other recombinant DNA molecules(s) and / or non-coding RNA (e.g., approximately 2.6 μg of DNA). Immediately after adding the DNA and / or RNA, add ice-cold sterile water to bring the final volume of the DNA and particle mixture to 245 μl. Immediately after bringing the mixture to this volume, add 250 μl of 2.5 M ice-cold CaCl2 solution and 50 μl of 0.1 M sterile spermidine. Mix this solution by slow vortex stirring. Incubate the tube on ice for at least 45 minutes to achieve particle coating. For better results in some experiments, mix the solution every 5–10 minutes. Pellet the particles by slow centrifugation (e.g., by using an Eppendorf 5815 microcentrifuge at 800–1000 rpm for 2 minutes). Wash the pellet with 1 ml of ethanol, wash the particles with a pipette tip, and pelletize by centrifugation. Remove the ethanol and add 36 μl of 100% ethanol, then resuspend the particles by stirring at a low speed vortex. Use 5 μl of this preparation for each impact with the helium particle gun. For electric guns (Accell), this preparation can be modified by combining 10 sets of 36 μl bead / particle preparations in a scintillation vial and adding 100% EtOH to yield a final volume of 20 ml.

[0116] The above sonication step can be performed at 45-55 kHz for 1 minute; the centrifugation step before bead coating can be performed in an IEC microcentrifuge at 5000 rpm (2300 g) for 10 seconds; and the centrifugation step after DNA coating of beads can be performed in an IEC microcentrifuge at 1000 rpm (100 g) for 2 minutes.

[0117] For particle impact, after three washes with sterile water, the desired amount of tungsten particles (Bio-Rad Laboratories) were resuspended in 50 μl of sterile water. Then, 2 μl of TransIT® 2020 (Mirus Bio LLC) and 30 μl of DNA and / or RNA prepared as provided above were added to the particles and gently mixed on ice for at least 10 minutes. The coated tungsten particles were then pelletized in a microcentrifuge at 8000 × g for 30 seconds, and the supernatant fraction was removed. The pellet was resuspended in 180 μl of sterile water by short-term sonication. Immediately after sonication, the coated particles were loaded onto 6 × macrocarriers (30 μl each) and air-dried for approximately 2-3 hours.

[0118] Example 2. Pre-culture of soybean explants for particle impact. The following is an example of a protocol for pre-culturing explants for impact. Dried excised soybean embryo explants are pre-culturised before particle impact. Mature embryo explants are excised from dried soybean seeds, for example, generally as described in U.S. Patent No. 8,362,317. The explants are weighed for spraying and rehydrated for 1 hour in either 20% PEG4000 (Lynx 3017; see, e.g., U.S. Patent Application Publication No. 2016 / 0264983) or 10% sucrose medium, and then thoroughly rinsed. Lynx 1595 medium (see, e.g., U.S. Patent Application Publication No. 2016 / 0264983) (or Lynx 1595 containing 30 ppm Cleary) may also be used in this step. Pre-culture approximately 50 explants per plate on EJW 1 or EJW 2 medium (see, for example, U.S. Patent Application Publication 2016 / 0264983). TDZ levels in the range of approximately 0.5 ppm to 2 ppm are also used in EJW (LIMS 4859) medium. Pre-culture the explants at 28°C for 1 to 2 days with either a 16 / 8-hour photoperiod or in the dark. Explants can also be pre-cultured for approximately 3 days.

[0119] Example 3. Particle impact on explants The following protocol can be used with the PDS1000 helium particle gun. Disinfect the gun components (stop screen, rupture disc, and macrocarrier holder, etc.) with 70% EtOH (or isopropanol for the carrier sheet) for approximately 1 minute. Load the rupture disc (e.g., a disc used in the range of approximately 650-2200 psi, e.g., a 1350 psi disc) into the rupture disc retaining cap and screw it into the gas acceleration chamber. Place the stop screen on the adjustable brass nest. Dispense 5 μl of the helium gun preparation described above onto each carrier sheet for each impact. Air dry the carrier sheets, then turn them over and place them on top of the stop screen or retaining screen on the brass nest. Assemble the macrocarrier launch assembly and place it directly under the rupture disc. The gap distance between the rupture disc and the macrocarrier launch assembly is approximately 1 cm.

[0120] Pre-cultured soybean explants are positioned on Target Plate Medium #42 (TPM42) with the center of the meristem facing upwards and sprayed. TPM42 medium is prepared by measuring 2 liters of distilled water into a 4 L beaker, adding 16 g of washed agar, and then autoclaving it for 25 minutes to dissolve the agar. TPM42 may contain 8% carboxymethylcellulose (CMC) for low viscosity (or 2% carboxymethylcellulose (CMC) for high viscosity) and 0.4% washed agar. The solution is slightly cooled and poured into a 4 L blender, and then 320 g of CMC (low viscosity) or 80 g of CMC (high viscosity) is added together with 2 L of water. The mixture is blended, transferred to a 4 L plastic beaker, then mixed, autoclaved for 30 minutes, and divided into four 1 L bottles. Next, autoclave the TPM42 solution for another 25 minutes, cool it to approximately 60°C, and then pour it into the plates. Approximately 300 target plates can be prepared by pouring about 12-15 ml per 60 mm plate, and these can be stored at 4°C or -20°C.

[0121] The following is an example of a protocol for using an ACCELL electric particle gun. Bring the bead preparation to room temperature and stir with a vortex. 3.2 cm at 0.5 mil 2 Place the Mylar sheet on a small plastic dish in a dehumidifying unit as desired, and place 320 μl of bead preparation on top of the sheet. Air dry each sheet. Position the pre-cultured soybean explants on a TPM42 plate with the center of the meristem facing upwards. Perform a blank spray first, as the energy of the initial spray is inconsistent. Place the target on a holding screen placed directly on the carrier sheet. Under partial helium vacuum (13.5 Hg), evaporate 10 μL of water droplets by discharging a capacitor at 17.5-20 kV. The shock wave formed by the evaporating droplets propels the sheet into the holding screen, which stops most of the Mylar but allows the gold beads to penetrate the soybean explant meristem. Between sprays, suspend a drop of mineral oil between the points, then remove and purify. Suspend 10 μL of water between the points as before. Cover the arc chamber with a PVC block, place a Mylar sheet over the square opening, and place the screen hood over the sheet and point. Align the screen over the sheet. With the meristem facing upwards, place the target dish upside down on the holding screen so that the weight is applied to the dish. Cover the apparatus with a bell-shaped jar and create a vacuum. After 15 seconds, read a vacuum of 13.5 Hg and fire the gun.

[0122] Example 4. Cultivation of explants following particle impact. The impacted explants are plated overnight on the surface of EJW 1 medium (other pre-culture media may also be used). In one example, the plates are incubated at 28°C with a 16 / 8-hour photoperiod. The explants are plated or embedded on the surface of B5 medium containing 50–500 ppm spectinomycin (LIMS 3485 with modified spectinomycin levels; see, e.g., U.S. Patent Application Publication 2016 / 0264983) and maintained at 28°C with a 16 / 8-hour photoperiod throughout the regeneration process. In one example, 250 ppm spectinomycin is used in the B5 medium. The presence of the aadA selectable marker gene provides resistance or tolerance to spectinomycin as a selective agent. 24.5 g of B5 custom medium mixture contains 3.21 g of Gamborg's B5 medium, 20 g of sucrose, and 1.29 g of calcium gluconate. The culture is monitored for bud / greening, and subculture is performed as needed.

[0123] Example 5. Identification of edited PDS mutations after particle delivery and regeneration. DNA fragments, shown in Figure 1B, corresponding to either 0.02 or 0.04 pmole per shot, were loaded onto particles for microparticle gun delivery. Plants regenerated from impacted explants were screened for the presence of the aadA marker. Plants positive for the aadA marker gene, as determined by real-time quantitative PCR, were further tested for the presence of editing resulting from co-delivery of recombinant DNA expressing Cas9 and gRNA targeting the PDS gene locus. Genomic DNA was extracted from leaf samples of small plants regenerated two weeks after impact, and the presence of editing at one or both PDS gene loci was detected by fragment length analysis (FLA). FLA is a PCR-based molecular analysis that identifies samples with one or more mutations compared to the wild-type reference by comparing the variation in PCR fragment length from a wild-type reference to an amplicon. PCR reactions were performed using 5' FAM-labeled primers, standard primers, and Phusion® polymerase (Thermo Fisher Scientific) according to the manufacturer's instructions to generate PCR fragments of 200–500 bp. The FLA primers for the GmPDS genes described in Sequence IDs 6 and 7 produce a 428 bp PCR fragment for GmPDS11 and a 384 bp PCR fragment for GmPDS18. PCR fragments of different sizes than those expected are considered to be mutated or edited alleles. As shown in Figure 1, the editing frequency was observed to range from 17.6% to 42.5% at the GmPDS locus after impact. [Table 1]

[0124] Example 6.18 Identification of site-directed integration (SDI) events into PDS loci on chromosome number. Delivering Cas9 and gRNA along with a donor template containing the insertion sequence can lead to site-directed integration (SDI) of the insertion sequence at the target site for the gRNA. In some cases, a DNA molecule or fragment encoding one or more genome editing components (e.g., site-specific nucleases, gRNAs, and / or marker genes) can also be used as a template for site-directed integration. Insertion of a DNA molecule or fragment can occur via non-homologous end joining (NHEJ) even if the DNA molecule or fragment does not contain homologous sequences for homology-mediated repair. Example 5 above demonstrates that multiple genome edits are produced via particle impact with the DNA fragment provided in Figure 1B. The DNA fragment in Figure 1B used to deliver the genome editing components in this example was also able to be inserted at the gRNA / Cas9 target cleavage site via NHEJ. In this experiment, two integration events were detected at the gRNA target site in the DNA fragment in Figure 1B.

[0125] Four PCR assays were established to detect the presence of insertion sequences, and the presence of insertion sequences could occur in two different orientations, as illustrated in Figure 1C. DNA fragments from Example 5 encoding Cas9 and gRNA2 (SEQ ID NO: 4) in either 0.02 or 0.04 pmole per shot were loaded into beads. One orientation of SDI insertion of the DNA fragment was detected using PCR with primers 3965 (SEQ ID NO: 8) and 2438 (SEQ ID NO: 9), and / or PCR with primers 4005 (SEQ ID NO: 10) and 3966 (SEQ ID NO: 11), as shown in Figure 1C, while the other orientation of SDI was detected using PCR with primers 3966 and 2438, and / or PCR with primers 4005 and 3965.

[0126] Events #48 and #20, which contained at least a portion of a DNA fragment insertion, were found by PCR (see Figure 2), and this was further confirmed by sequencing via the integration junction. Event #20 had an intact NotI sequence at the insertion end, but the other I-CeuI end of the fragment was not detected by PCR, suggesting that a deletion or structural change that prevented PCR amplification occurred at this end. Event #20 occurred at the PDS locus on chromosome 18, and Taqman analysis further revealed that all or part of more than four copies of the DNA fragment were integrated at that site. Event #48 was determined to have a deletion at the insertion end (I-CeuI end) detected by PCR with primers 3966 and 4005. The other end of the insertion in event #48 was not detected by PCR, suggesting that a deletion or structural change that prevented PCR amplification occurred at this end. Similar to event #20, event #48 occurred at the PDS locus on chromosome 18, and Taqman analysis further revealed that all or part of more than four copies of a DNA fragment were incorporated at that site.

[0127] The number and frequency of SDI events at the GmPDS(Chr18) locus are provided in Table 2. Mutations in the PDS gene can cause a whitened leaf phenotype, which may indicate that editing of the PDS gene has occurred and / or that such editing is present in those tissues. [Table 2]

[0128] Example 7. Delivery of Cpf1, gRNA, and ssDNA into mature seed explants. Since LbCpf1 shows preference for TTTV PAM sequences, the target site GmTS1 was selected based on the appearance of appropriate PAM sequences upstream of each target sequence. A guide RNA was designed to guide the LbCpf1 protein to the target site. Recombinant DNA for expressing the LbCpf1 protein, guide RNA, and aadA was cloned.

[0129] Furthermore, a 70 bp long ssDNA (single-stranded DNA) template with 5'TEG modification was designed. The TEG-modified ssDNA template was obtained from Integrated DNA Technologies (IDT, product 1184, modification code: / 5Sp9 / ). This template has a 10 bp signature sequence containing a BamHI recognition sequence adjacent to a 30 bp 5' homologous arm and a 30 bp 3' homologous arm (designed to be identical to the DNA sequence adjacent to the GmTS1 site). The corresponding wild-type sequence at the GnTS1 site has an 8 bp endogenous sequence between the 5' and 3' homologous arms. This single-stranded DNA template (ssDNA template) was added to recombinant DNA for the expression of LbCpf1 protein, crRNAs, and aadA. Specifically, 80 pmol of ssDNA template and 0.8 pmol of recombinant DNA were coated onto 0.6 μm gold particles (Bio-Rad; approximately 66 μg / shot) using TransIT-2020 as the coating reagent. The mixture was maintained on ice for ≥15 minutes, gently mixed every 5 minutes. The coated gold particles were pelletized by short-term centrifugation, and the supernatant was removed. The coated gold particles were resuspended and washed with 1 mL of pre-cooled 100% ethanol, followed by short-term centrifugation to remove the ethanol. The coated gold particles were resuspended in 30 μl of pre-cooled 100% ethanol, then packed onto a microcarrier disk (5 μl per shot), and dried for 10 minutes.

[0130] Dried excised soybean embryo explants were rehydrated for 1 hour in LIMS 3990 (30 g / L B5 custom medium containing 1 g / L KNO3, 0.03 g / L Clearys 3336 WP, and 3.9 g / L MES (pH to 5.6)), thoroughly rinsed with sterile H2O, and cultured in LIMS 4859 medium at 28°C with a 16 / 8 hour photoperiod for 1 day. These pre-cultured mature soybean embryo explants were subjected to shock according to Example 3. After shock, the embryo explants were transferred onto LIMS 4859 medium and cultured in the dark at 28°C or 37°C for 2 days.

[0131] As shown in Table 3, over 15 experiments at 28°C, the mutation rate (small insertions or deletions around the designed Cpf1 cleavage site) was 41.7%, and the perfect template editing rate (having a 10bp signature sequence containing a BamHI recognition sequence with adjacent 5' and 3' junctions defined by homologous arms) was 0.25%. Over 15 experiments at 37°C, the mutation rate (small insertions or deletions) was 49.9%, and the perfect template editing rate (having a 10bp signature sequence containing a BamHI recognition sequence with adjacent 5' and 3' junctions defined by homologous arms) was 0.35%. [Table 3]

[0132] While the present invention has been disclosed with reference to certain embodiments, it will be apparent that modifications and variations are possible without departing from the spirit and scope of the invention as disclosed herein and provided by the appended claims. Furthermore, it should be recognized that all embodiments in this disclosure, while illustrating embodiments of the invention, are provided as non-limiting embodiments and should not be considered as limiting the various embodiments thus illustrated. All references cited herein are incorporated herein by reference to their entirety. The present invention is intended to have the full scope defined by this disclosure, the wording of the following claims, and any equivalents thereof. Accordingly, the drawings and embodiments for carrying out the invention should be considered illustrative and not limiting.

Claims

1. A method for editing the genome of a plant, a) Extracting a mature embryo from a dried, mature plant seed, thereby producing an excised mature plant embryo explant; b) Delivering a recombinant DNA construct, comprising a sequence encoding a site-specific nuclease, wherein the sequence is operably linked to a plant-expressible promoter, to the excised mature plant embryo explant via particle impact; c) The excised mature plant embryo explant is cultured at approximately 37°C after impact; d) Regenerating a plant from the excised mature plant embryo explant without forming an embryogenetic callus or callus culture from the excised mature plant embryo explant, wherein the regenerated plant includes editing or site-directed integration of the site-directed nuclease at or near the target site in the genome of at least one cell of the regenerated plant; and e) Identifying regenerated plants having at least one cell containing the editing or site-directed integration at or near the target site of the site-specific nuclease, based on phenotype or trait, or based on molecular assays. The method, including the method described above.

2. The method according to claim 1, wherein the particles coated with the recombinant DNA construct are delivered to the excised mature plant embryo explant via particle impact.

3. The method according to claim 2, wherein the particles are tungsten particles, platinum particles, or gold particles.

4. The method according to claim 2 or 3, wherein the particles have a size between 0.5 μm and 1.5 μm.

5. The method according to claim 4, wherein the particles have a size of 0.6 μm, 0.7 μm, or 1.3 μm.

6. The method according to claim 2, wherein a plurality of particles coated with the recombinant DNA molecule are delivered to the excised mature plant embryo explant via particle impact.

7. The method according to claim 6, wherein the amount of particles delivered to the explant is between 50 μg and 5000 μg.

8. The method according to any one of claims 1 to 7, wherein the site-specific nuclease is a ribonucleoprotein.

9. The method according to any one of claims 2 to 7, wherein the particles are further coated with guide RNA.

10. The aforementioned site-specific nucleases are Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Csn1, Csx12, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cm The method according to any one of claims 1 to 9, wherein r5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, CasZ, or an Argonaut protein, or a homolog or modified version thereof.

11. The method according to any one of claims 1 to 9, wherein the site-specific nuclease is Cas9 protein.

12. The method according to claim 11, wherein the Cas9 protein is derived from Streptococcus pyogenes.

13. The method according to any one of claims 1 to 9, wherein the site-specific nuclease is the Cpf1 protein.

14. The method according to any one of claims 1 to 7, wherein the site-specific nuclease is not a guide-type nuclease.

15. The method according to claim 14, wherein the site-specific nuclease is a meganuclease, a zinc finger nuclease (ZFN), a recombinase, a transposase, or a transcription activator-like effector nuclease (TALEN).

16. The method according to any one of claims 1 to 15, wherein the delivery step further comprises delivering a second recombinant DNA construct or molecule to the excised mature plant embryo explant.

17. The method according to any one of claims 2 to 7, wherein the particles are further coated with a second recombinant DNA construct or molecule.

18. The method according to claim 16 or 17, wherein the second recombinant DNA construct or molecule is a donor template.

19. The method according to claim 18, wherein the donor template comprises a homologous sequence containing the mutation for introducing the mutation into the genome of the plant via template-mediated repair at or near the target site of the site-specific nuclease.

20. The method according to claim 18, wherein the donor template includes the insertion sequence and at least one homologous sequence for incorporating the insertion sequence into the genome of the plant at or near the target site of the site-specific nuclease.

21. The method according to claim 20, wherein the inserted sequence comprises a transgene including a coding sequence or a transcriptional DNA sequence operably linked to a promoter expressible in a plant.

22. The method according to claim 21, wherein the introduced gene includes a gene of interest.

23. The method according to claim 21, wherein the introduced gene includes a protein coding sequence.

24. The method according to claim 21, wherein the introduced gene comprises a transcribed DNA sequence encoding a non-coding RNA molecule.

25. The method according to claim 21, wherein the introduced gene includes a marker gene.

26. The method according to claim 16 or 17, wherein the second recombinant DNA molecule comprises a marker gene.

27. The method according to claim 26, wherein the marker gene is a selectable marker gene.

28. The method according to claim 27, wherein the selectable marker gene includes an adenylyltransferase (aadA) gene, a neomycin phosphotransferase (nptII) gene, a hygromycin phosphotransferase (hpt, hph, or aph IV), a 5-enoylpyruvinylshikimic acid-3-phosphate synthase (EPPSPS) gene, or a dicamba monooxygenase (DMO) gene, or a bialaphos resistance (bar) or phosphinotricin N-acetyltransferase (pat) gene.

29. The method according to claim 27, wherein the selectable marker gene includes the adenylyltransferase (aadA) gene.

30. The method according to claim 26, wherein the marker gene is a screenable marker gene.

31. The method according to claim 30, wherein the screenable marker gene comprises a green fluorescent protein (GFP) gene or a β-glucuronidase (GUS) gene.

32. The method according to claim 16 or 17, wherein the second recombinant DNA construct or molecule comprises a donor template region and a transgene comprising a coding sequence or a transcriptionable DNA sequence, and the transgene is located outside the donor template region of the second recombinant DNA construct or molecule.

33. The method according to claim 16 or 17, wherein the second recombinant DNA construct or molecule comprises a transcriptionable DNA sequence encoding a guide RNA, and the transcriptionable DNA sequence is operably linked to a promoter expressible in a plant.

34. The method according to any one of claims 1 to 33, wherein the recombinant DNA construct further comprises a marker gene.

35. The method according to claim 34, wherein the marker gene is a selectable marker gene.

36. The method according to claim 35, wherein the selectable marker gene includes an adenylyltransferase (aadA) gene, a neomycin phosphotransferase (nptII) gene, a hygromycin phosphotransferase (hpt, hph, or aph IV), a 5-enoylpyruvinylshikimic acid-3-phosphate synthase (EPPSPS) gene, or a dicamba monooxygenase (DMO) gene, or a bialaphos resistance (bar) or phosphinotricin N-acetyltransferase (pat) gene.

37. The method according to claim 35, wherein the selectable marker gene includes the adenylyltransferase (aadA) gene.

38. The method according to claim 34, wherein the marker gene is a screenable marker gene.

39. The method according to claim 38, wherein the screenable marker gene comprises a green fluorescent protein (GFP) gene or a β-glucuronidase (GUS) gene.

40. The method according to any one of claims 1 to 39, wherein the recombinant DNA construct further comprises a transcriptionable DNA sequence encoding a guide RNA, and the transcriptionable DNA sequence is operably linked to a promoter expressible in a second plant.

41. The method according to any one of claims 1 to 39, wherein the recombinant DNA construct further comprises a donor template region.

42. The method according to claim 41, wherein the donor template region comprises a homologous sequence containing the mutation for introducing the mutation into the genome of the plant via template-mediated repair at or near the target site of the site-specific nuclease.

43. The method according to claim 41, wherein the donor template region includes the insertion sequence and at least one homologous sequence for incorporating the insertion sequence into the genome of the plant at or near the target site of the site-specific nuclease.

44. The method according to claim 43, wherein the inserted sequence comprises a transgene including a coding sequence or a transcriptional DNA sequence operably linked to a promoter expressible in a plant.

45. The method according to claim 44, wherein the introduced gene includes a gene of interest.

46. The method according to claim 44, wherein the introduced gene includes a protein coding sequence.

47. The method according to claim 44, wherein the introduced gene comprises a transcriptable DNA sequence encoding a non-coding RNA molecule.

48. The method according to claim 44, wherein the introduced gene includes a marker gene.

49. f) Select a regenerated plant having a marker gene, and the marker gene is co-delivered together with the recombinant DNA molecule. The method according to any one of claims 1 to 48, further comprising:

50. The method according to claim 49, wherein the marker gene is a selectable marker gene.

51. The method according to claim 50, wherein the selection step comprises treating the mature embryo explant, or the culture of buds and / or roots, or the vegetative plants regenerated therefrom, with a selective agent.

52. The method according to claim 50, wherein the selectable marker gene is the adenylyltransferase (aadA) gene.

53. The method according to any one of claims 1 to 52, wherein the plant is a dicotyledonous plant.

54. The method according to claim 53, wherein the plant is a soybean plant.

55. The method according to any one of claims 1 to 54, wherein the mature embryo explant comprises, prior to the delivery step, one or more of the following: (i) guide RNA (gRNA), (ii) a polynucleotide comprising a transgene or marker gene, (iii) a polynucleotide comprising a non-coding RNA molecule or a transgene encoding a guide RNA, and / or (iv) a donor template.

56. The method according to any one of claims 1 to 55, wherein the mature embryo explant has a water content in the range of 3% to 25%.

57. The method according to any one of claims 1 to 56, wherein the mature embryo explant is excised from a plant seed having a water content in the range of 3% to 25%.

58. The method according to claim 16 or 17, wherein the delivery step comprises delivering a DNA molecule or vector comprising the recombinant DNA construct and the second recombinant DNA construct to the mature plant embryo explant.

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