Plant genome editing

Abstract

Compositions for genome editing and site-specific integration in plants are provided comprising microprojectile particles coated, treated or applied with a recombinant DNA construct for delivery to a mature embryo explant from a dry seed, the DNA construct comprising a sequence encoding one or more genome editing repeat sequences. Methods of using the disclosed compositions for genome editing and site-specific integration in at least one cell of a plant, and plants, plant parts and seeds comprising an edited genome or site-specific integration produced by the disclosed methods are also provided.

Description

[0001] This application is a divisional application of the invention patent application filed on May 24, 2019, with application number 201980029517.4 and title "Plant Genome Editing". Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 676,228, filed May 24, 2018, which is incorporated herein by reference in its entirety.

[0003] Merging of sequence lists The sequence list contained in the file named “MONS444WO_ST25.txt” is submitted electronically and incorporated herein by reference. The size of the sequence list is 4 kilobytes as measured in the Microsoft Windows operating system and was created on May 23, 2019. Technical Field

[0004] This disclosure relates to compositions for genome encoding in plants using DNA molecules that encode genome editing reagents, and methods of using such compositions. Background Technology

[0005] Precise genome editing technologies hold promise as powerful tools for engineering gene expression and function, with the potential to improve agriculture. There is an ongoing need in this field to develop novel compositions and methods for effectively and efficiently editing plant genomes. Summary of the Invention

[0006] This disclosure provides a method for editing a plant genome, comprising: delivering a recombinant DNA construct to a mature plant embryo explant, the recombinant DNA construct comprising a sequence encoding a site-specific nuclease, wherein the sequence is operatively linked to a plant-expressible promoter; and regenerating a plant from the mature plant embryo explant, wherein in the genome of at least one cell of the regenerated plant, the regenerated plant contains editing or site-specific integration at or near a target site of the site-specific nuclease. In some 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 via Agrobacterium (…). Agrobacterium T-DNA transformation vectors containing recombinant DNA constructs are delivered to mature plant embryo explants via mediated transformation. In some embodiments, a T-DNA transformation vector is delivered to mature plant embryo explants.

[0007] In another embodiment, the recombinant DNA construct is delivered to a mature plant embryo explant via particle bombardment. In some embodiments, particles coated or applied with the recombinant DNA construct are delivered to the mature plant embryo explant via particle bombardment. In some embodiments, the particles are tungsten, platinum, or gold particles. In other embodiments, the particle size is between about 0.5 µm and about 1.5 µm. In other embodiments, the particle size is about 0.6 μm, about 0.7 μm, or about 1.3 μm. In some embodiments, multiple particles coated or applied with recombinant DNA molecules are delivered to the mature plant embryo explant via particle bombardment. In some embodiments, the amount of particles delivered to the explant is between about 50 µg and about 5000 µg, or between about 50 µg and about 5000 µg, or between about 50 µg and about 2000 µg, or between about 50 µg and about 1000 µg, or between about 50 µg and about 500 µg, or between about 100 µg and about 500 µg.

[0008] In some embodiments, the method further includes identifying regenerated plants having cells containing edited or site-specifically integrated cells at or near a target site of a site-specific nuclease. In some embodiments, the identification step includes identifying regenerated plants with edited or site-specific integration based on phenotype or trait. In other embodiments, the identification step includes identifying regenerated plants with edited or site-specific integration based on molecular assays.

[0009] In another embodiment, the site-specific nuclease is a guide nuclease, such as a CRISPR-associated protein. In other embodiments, the particle is further coated or coated with a guide nucleic acid. In some embodiments, the guide nuclease is 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, Csm 6. Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, CasZ, or Argonaute protein, or their homologs or modified forms. In other embodiments, the guiding nuclease is the Cas9 protein. In other embodiments, the Cas9 protein is derived from *Streptococcus pyogenes* (…). Streptococcus pyogenesIn another embodiment, the guiding nuclease is the Cpf1 protein. In some embodiments, the site-specific nuclease is not a guiding nuclease. In other embodiments, the site-specific nuclease is a meganuclease, a zinc finger nuclease (ZFN), a recombinase, a transposase, or a transcription activator-like effector nuclease (TALEN).

[0010] In some embodiments, the delivery step further includes delivering a second recombinant DNA construct or molecule to a mature plant embryo explant. In some embodiments, the particle is further coated or applied with the second recombinant DNA construct or molecule. In other embodiments, the second recombinant DNA construct or molecule serves as a donor template. In other embodiments, the donor template contains a homologous sequence containing a mutation for introducing the mutation into the plant genome at or near the target site of a site-specific nuclease via template-mediated repair. In other embodiments, the donor template contains an insert sequence and at least one homologous sequence for integrating the insert sequence into the plant genome at or near the target site of a site-specific nuclease.

[0011] In certain embodiments, the insert sequence comprises a transgene containing a coding sequence or transcribed DNA sequence operatively linked to a plant-expressible promoter. In some 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 other embodiments, the transgene contains a marker gene. In yet another embodiment, the second recombinant DNA molecule contains a marker gene. In some embodiments, the marker gene is a selection marker gene. In some embodiments, the selection marker gene contains an adenylate transferase (…). aadA ) gene, neomycin phosphotransferase ( nptII ) gene, hygromycin phosphotransferase ( hpt , hp or aph IV ), 5-enolpyruvate shikimate-3-phosphate synthase ( EPSPS ) gene, dicamba monooxygenase (DMO) gene or diammonium phosphate resistance ( bar ) or glufosinate N-acetyltransferase ( pat In another embodiment, the selection marker gene includes adenylate transferase (ALT). aadA In another embodiment, the marker gene is a selection marker gene. In various embodiments, the selection marker gene comprises a green fluorescent protein (GFP) or β-glucuronidase (GUS) gene.

[0012] In another embodiment, the second recombinant DNA construct or molecule comprises a donor template region and a transgene comprising a coding sequence or a transcribed DNA sequence, wherein the transgene is 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 transcribed DNA sequence encoding a guide nucleic acid, wherein the transcribed DNA sequence is operatively linked to a plant-expressible promoter. In some embodiments, the recombinant DNA construct further comprises a marker gene. In some embodiments, the marker gene is a selection marker gene. In various embodiments, the selection marker gene comprises adenylate transferase (…). aadA ) gene, neomycin phosphotransferase ( nptII ) gene, hygromycin phosphotransferase ( hpt , hp or aph IV ), 5-enolpyruvate shikimate-3-phosphate synthase ( EPSPS ) gene, dicamba monooxygenase (DMO) gene or diammonium phosphate resistance ( bar ) or glufosinate N-acetyltransferase ( pat In another embodiment, the selection marker gene includes adenylate transferase (ALT). aadA In other embodiments, the marker gene is a selection marker gene. In other embodiments, the selection marker gene comprises a green fluorescent protein (GFP) or β-glucuronidase (GUS) gene.

[0013] In some embodiments, the recombinant DNA construct further comprises a transcribed DNA sequence encoding a guide nucleic acid, wherein the transcribed DNA sequence is operatively linked to a second plant-expressible promoter. In other embodiments, the recombinant DNA construct further comprises a donor template region. In some embodiments, the donor template region comprises a homologous sequence containing a mutation for introducing the mutation into the plant genome at or near the target site of a site-specific nuclease via template-mediated repair. In other embodiments, the donor template region comprises an insert sequence and at least one homologous sequence for integrating the insert sequence into the plant genome at or near the target site of a site-specific nuclease. In other embodiments, the insert sequence comprises a transgene containing a coding sequence or a transcribed DNA sequence operatively linked to a plant-expressible promoter. In further embodiments, the transgene comprises a gene of interest. In a particular embodiment, the transgene comprises a protein-coding sequence. In some embodiments, the transgene comprises a transcribed DNA sequence encoding a non-coding RNA molecule. In some embodiments, the transgene comprises a marker gene.

[0014] In another embodiment, the method further includes selecting regenerated plants with a marker gene, wherein the marker gene is co-delivered with a recombinant DNA molecule. In some embodiments, the marker gene is a selection marker gene. In other embodiments, the selection step includes treating mature embryonic explants, or shoot and / or root cultures or plantlets regenerated from them, with a selection agent. In some embodiments, the selection marker gene is an adenylate transferase (ALT). aadA )Gene.

[0015] Several embodiments relate to a method for editing a plant genome, comprising: a) delivering a recombinant DNA construct to a mature plant embryo explant, the recombinant DNA construct comprising a sequence encoding one or more genome editing agents, wherein the sequence is operatively linked to a plant-expressible promoter; and b) regenerating a plant from the mature plant embryo explant, wherein in the genome of at least one cell of the regenerated plant, the regenerated plant contains editing or site-directed integration at or near a target site. In some embodiments, the method further comprises identifying regenerated plants 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 regenerated plants having a marker gene, wherein the marker gene is 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, the recombinant DNA construct is delivered to the mature plant embryo explant via particle bombardment. In some embodiments, the particles are tungsten, platinum, or gold particles. In some embodiments, the particle size is 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 about 1.5 μm. In some embodiments, the particles are further coated or coated with a second recombinant DNA construct or molecule. In some embodiments, the second recombinant DNA construct or molecule is a donor template.

[0016] In some embodiments, the plant is a dicotyledonous plant. In a particular embodiment, the plant is a soybean plant. In other embodiments, the plant is a monocotyledonous plant. In yet another embodiment, the mature embryo explant comprises one or more of the following prior to the delivery step: (i) a guide nucleic acid, (ii) a polynucleotide containing a transgenic or marker gene, (iii) a polynucleotide containing a transgenic gene encoding a non-coding RNA molecule or a guide nucleic acid, and / or (iv) a donor template. In some embodiments, the mature embryo explant is a dry-cut explant. In other embodiments, the mature embryo explant is a wet embryo explant, a dry wet embryo explant, or a wet-cut embryo explant. In other embodiments, the moisture content of the mature embryo explant is in the range of about 3% to about 25%. In other embodiments, the mature embryo explant is excised from plant seeds with a moisture content in the range of about 3% to about 25%. In yet another embodiment, the delivery step comprises delivering a DNA molecule or vector containing the recombinant DNA construct and the second recombinant DNA construct to the mature plant embryo explant. Attached Figure Description

[0017] The following figures form part of this specification and are included to further illustrate certain aspects of this disclosure. This disclosure can be better understood by referring to one or more of these figures in conjunction with the detailed description of the specific embodiments presented herein.

[0018] Figure 1A A diagram showing the two PDS loci on chromosomes 11 (Ch11) and 18 (Ch18) in soybean, which shows the target sites of three guide RNAs.

[0019] Figure 1B To display a diagram of the NotI / I-CeuI restrictive fragment, the restrictive fragment is cut off from the part having the function of... just and aadA A vector for the expression cassette of marker genes, Cas9 nuclease, and guide RNA, and its use for particle bombardment of explants to generate genome editing at target sites within the PDS gene locus.

[0020] Figure 1C To illustrate the different possible orientations for inserting the complete NotI / I-CeuI restriction fragment into the PDS gene locus, the binding sites of the PCR primers (3965, 3966, 2438, and 4005) for each orientation are shown.

[0021] Figure 2To show gel images of PCR products generated using different primer pairs (3965 and 4005, or 3966 and 4005), which show two site-directed integration (SDI) events (event #20 and event #48) in the edited sample being tested. Detailed Implementation

[0022] Gene function analysis and crop improvement using genome editing technologies hold great promise for agricultural improvement. Although genome editing reagents have been delivered in the form of DNA to culturable plant cells, the delivery of DNA encoding genome editing reagents to mature plant embryo explants to induce regeneration or development of edited plants has not been described. The regenerative capacity of plant cells and tissues transformed with DNA encoding genome editing reagents may be limited, and many plant germplasms may not be suitable for these culture methods. In fact, many crop plants and varieties cannot effectively form callus, suspension cultures, or protoplasts. Therefore, existing genome editing methods can be species- and genotype-dependent depending on the type of explant and culture requirements, and in many cases limited to commercially less viable germplasms and agronomically important crop varieties that require more recurrent crosses with more superior donor systems to infiltrate genome editing or site-directed integration into the desired genetic background.

[0023] This disclosure overcomes the limitations of the art by providing a method for delivering genome editing agents in the form of DNA encoding genome editing agents (such as nuclease proteins or guide nucleic acids) to mature or dried explants (DEEs), said DNA being delivered via bacterial-mediated transformation or by coating onto particles for bio-projectile delivery to the explants. "Dried explant" refers to a mature embryonic explant obtained or excised from mature dried seeds. Plant seeds are naturally dried during their maturation process. As further described below, other types of explants may be obtained or excised from mature seeds, depending on the treatment method, such as after wetting, imbibing, etc., of the dried seeds, and the explants may be wetting, imbibing, etc., after excising from the dried seeds. Editing or site-specific integration can be achieved by delivering DNA encoding genome editing agents (such as nuclease proteins or guide nucleic acids) to one or more cells of meristematic tissue (such as embryonic meristem) without any prior callus formation steps. By avoiding the need for a callus phase prior to the delivery of the genome editing reagent to one or more cells of the target explant according to the method of the present invention, genotype and species dependence on existing methods can be reduced or eliminated, and efficient delivery of DNA encoding genome editing reagents (such as nuclease proteins or guide nucleic acids) to those target explants can be achieved. Therefore, the currently disclosed methods for delivering genome editing reagents can be carried out directly in a variety of plant germplasms, including superior germplasm lines of agronomically important crop species, thereby allowing direct regeneration of plants from desired germplasm containing inserted sequences or transgenes for targeted editing or site-directed integration.

[0024] The genome editing methods of this disclosure can be used to generate explants with desired editing or site-specific integration of sequences or transgenes, thereby allowing them to develop fairly normally into adult R0 plants containing the desired editing or site-specific integration with only a few culture and / or regeneration steps. Such R0 plants can develop or regenerate from the explants without requiring embryogenic or callus cultures. The targeted editing or site-specific integration in the R0 plants generated by the methods of this disclosure can be further disseminated in germlines to produce genome-edited R1 seeds and plants, as well as progeny with seeds and plants containing the desired editing or site-specific integration.

[0025] The ability to generate genome-edited R0 plants without the need for large-scale culturing of dry explants prior to the introduction of genome editing reagents (such as nuclease proteins or guide nucleic acids) allows for a faster and more efficient implementation of the methods disclosed herein, thus making them potentially suitable for large-scale commercial production of genome-edited crop plants. Dry explants can be obtained from seeds and used almost directly as targets for genome editing or site-directed integration. According to some embodiments, dry explants can be obtained from mature dry seeds and used as targets for editing that may require only minimal wetting, hydration, or pre-culturing steps. Therefore, dry explants from storable dry seeds can be conveniently used as targets for genome editing or site-directed integration of insert sequences or transgenes. As an alternative to dry explants, “wet” or “dry-wet” embryonic explants (including, for example, primed or germinating embryonic explants) can be used as targets for genome editing. Such “wet” embryonic explants are dry explants that have undergone wetting, hydration, imbibition, or other minimal culturing steps before receiving editing enzymes. Similarly, “wet explants” derived from imbibition or hydrated seeds can also be used as targets. “Wet” embryonic explants are hydrated or imbibition after being removed from the seed, while “wet explants” are removed from seeds that have already been hydrated or imbibition.

[0026] I. Conversion Methods Embodiments of this disclosure provide a method for editing the genome of dry explants (DEEs) derived from plant dry seeds, comprising delivering DNA encoding a genome editing agent (such as a nuclease protein or a guide nucleic acid) into at least one cell of the explant to produce genome editing or site-directed integration in at least one cell of the explant, said DNA being provided together with a donor template (for transformation) such as via bioprojectile particle-mediated or bacterial-mediated (e.g., Agrobacterium-mediated) delivery. The method of this disclosure can be performed by targeting dry explants harvested from seeds without extensive culture of the explants, followed by delivery of DNA encoding one or more genome editing agents. Such explants may be excised from storable dry seeds or may be “wet” embryo explants, “dry-wet” embryo explants, or “wet-excised” embryo explants.

[0027] According to some embodiments, dry explants excised from plant seeds may optionally be pre-cultured for a limited time in an aqueous medium before the DNA encoding genome editing reagents (such as nuclease proteins or guide nucleic acids) is delivered to the explants. This pre-culture medium may contain various salts (e.g., MS basal salts, B5 salts, etc.) and other components (such as various osmotic agents, sugars, antimicrobial agents, etc.). The pre-culture medium may be solid or liquid and may also contain one or more plant growth regulators or plant hormones, including one or more auxins, cytokinins, etc. According to some embodiments, multiple explants may be pre-cultured together in the same medium or container. For example, 2 to 100 explants (such as about 25, 50, 75, or 100 explants) may be plated on or in the same pre-culture medium, but larger numbers of explants may be pre-cultured together depending on the type of explant, the container, the size of the dish, etc. According to some embodiments, the pre-culture medium may contain: auxins, such as 2,4-D, indoleacetic acid (IAA), dicamba, 1-naphthaleneacetic acid (NAA), etc.; and cytokinins or similar growth regulators, such as thidiazuron (TDZ), 6-benzylaminopurine (BAP), zeatin, or zeatin nucleoside, etc. This pre-culture or pre-culture step can enhance the ability to edit and / or regenerate explants. The relative amounts of auxins and cytokinins (or similar growth regulators) in the pre-culture medium can be controlled or predetermined to enable successful modified editing and / or regeneration while preventing callus formation in explants (even over extended periods). According to some embodiments, the pre-culture medium may contain auxins (such as 2,4-D) and cytokinins (such as TDZ). For example, the concentration of cytokinins (such as TDZ) in the pre-culture medium (if present) can range from zero (0) to about 5 ppm, such as about 0.3 to about 4 parts per million (ppm), about 0.5 to about 3 ppm, about 1 to about 2, or in any other intermediate concentration range. In some respects, 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 some respects, the concentration of TDZ is 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 1.1 ppm, about 1.2 ppm, about 1.3 ppm, about 1.4 ppm, about 1.5 ppm, about 1.6 ppm, about 1.7 ppm, about 1.8 ppm, or about 1.9 ppm. The concentration of auxin (such as 2,4-D) can range from zero (0) to about 2 ppm, or from about 0.1 ppm to about 1 ppm, or from about 0.1 ppm to about 0.5 ppm, or in any other intermediate concentration range. In some respects, the concentration of auxin is 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 1.1 ppm, about 1.2 ppm, about 1.3 ppm, about 1.4 ppm, about 1.5 ppm, about 1.6 ppm, about 1.7 ppm, about 1.8 ppm, or about 1.9 ppm.

[0028] The duration of the pre-culture step may vary to some extent depending on the temperature of the pre-culture medium and / or the environment surrounding the explants. Typically, the pre-culture step time can also be controlled and limited to a range of approximately 1 or 2 hours to approximately 5 days, such as approximately 12 hours to approximately 60 hours, or approximately 12 hours to approximately 48 hours, or any other time range therein. Limiting the pre-culture step time, even with plant growth regulators, can also prevent callus formation. Optimal pre-culture time can also improve plant regeneration frequency. During the pre-culture step, explants can be kept on the same medium or transferred once or multiple times to one or more fresh media. The lighting and / or temperature conditions for the optional pre-culture step can also be controlled. For example, explants may be exposed to a 16 / 8 hour photoperiod during the pre-culture step, or possibly to various other light and dark cycles or times. Alternatively, the pre-culture step can be performed under dark or low-light conditions. The temperature variations of the explant pre-culture medium and surrounding environment can also be approximately 18°C ​​to approximately 35°C, or approximately 25°C to approximately 30°C, or approximately 28°C, and include all intermediate ranges and values.

[0029] According to some implementation schemes, and regardless of whether a pre-culture step is performed, the dry explants intended for transformation may optionally be exposed to a hydration or imbibition medium for a limited time prior to pre-culture and / or exposure to genome editing reagents. This hydration or imbibition step makes explants from dry seeds or desiccated seeds more suitable for editing or site-specific integration. In practice, a hydration or imbibition step can be performed prior to transformation without a separate pre-culture step. The hydration medium may consist of water only, or may also contain one or more known osmolarizing agents, such as one or more sugars (e.g., sucrose), polyethylene glycol (PEG), etc. For example, the hydration medium may include about 10% sucrose and / or about 20% PEG. Without being bound by any theory, osmolarizing agents can modulate or slow down the hydration rate of the explants. Other components, such as various salts, may also be included in the hydration medium. The duration of the hydration step can typically be short, such as about 2 minutes to about 12 hours, or about 20 minutes to about 6 hours, or about 30 minutes to about 2 hours, or about 1 hour. The hydration or imbibition step can be short enough that no germination or at least no observable germination or developmental changes occur in the explants. Alternatively, embryonic explants can be induced for germination, or even allowed to germinate before delivery of genome editing reagents. For example, embryonic explants can be induced for germination by wetting and then drying germination-arrested explants (to produce “dry-wet” embryonic explants). Furthermore, “wet-excised” embryos (embryo explants excised from hydrated or wet seeds) can also be used as targets for transformation. Various rinsing steps may also be performed before, during, and / or after any hydration and / or pre-culture steps.

[0030] According to some implementations, one or more hydration and / or pre-culture steps may be included prior to transformation to improve editing, particularly for dry (or dehydrated) explants, such as those obtained from mature and / or dry (or dehydrated) seeds. However, one or both of these steps may be optional depending on the moisture content and / or type of the explant used as the editing target. Nevertheless, hydration and / or pre-culture steps may be optional and may be omitted or not performed, particularly when “wet” or “wet-excised” embryonic explants are used as targets, as these explants may already have sufficient hydration levels or moisture content.

[0031] Regardless of whether the one or more hydration and / or pre-culture steps are performed, explants can be transformed with DNA molecules encoding genome editing agents (such as nuclease proteins or guide nucleic acids) to produce explants with at least one genome-edited cell. The DNA may be coated onto the particle for delivery as a biological projectile or incorporated into a vector (e.g., a T-DNA vector) for transformation. After delivery or transformation of the DNA encoding the genome editing agent, the explant can then be grown, developed, regenerated, etc., into a plant under selective pressure to select for the growth and development of one or more genome-edited cells. In some embodiments, the DNA encoding one or more genome editing agents may be co-transformed or co-delivered with a selection marker gene, such that the survival, growth, and development of the genome-edited cells are advantageous in the presence of a corresponding selector. According to some embodiments, the DNA encoding one or more genome editing agents may be co-transformed or co-delivered with a donor template molecule for site-specific integration of an insertion sequence or transgene (e.g., a gene of interest) into the plant cell genome. The donor template molecule for site-specific integration may also contain a selection marker gene that can be used as a basis for selection. According to some embodiments, the DNA encoding one or more genome editing agents may be co-transformed or co-delivered with a guide nucleic acid or DNA encoding a guide nucleic acid. According to some embodiments, the DNA encoding one or more genome editing agents transformed or delivered to the explant may also contain one or more of the following: a donor template sequence and / or a selection marker gene.

[0032] According to certain embodiments of this disclosure, a transformation vector incorporating DNA encoding one or more genome editing agents, or particles having, coated, or applied said DNA on their surface, are introduced into at least one cell of a target explant via explant transformation or particle-mediated bombardment. Such particle-mediated bombardment can utilize any suitable particle gun device known in the art, such as a helium particle gun, an electron particle gun, etc. Prior to bombardment, the particles may be loaded, applied, or coated with a copy of a DNA construct or molecule encoding one or more genome editing agents, and optionally a guide RNA, a marker gene, and / or a donor template. The DNA construct or molecule encoding one or more genome editing agents may contain sequences encoding guide RNA, a marker gene, and / or a donor template. The particles themselves may comprise any suitable type of particle or bead known in the art, such as gold or tungsten beads. The blasting conditions of the particle gun are well known in the art, and various conventional screens, rupture discs, etc., can be used, such as for a helium particle gun. Electron guns may offer advantages in reducing the amount of time required for transformation and in using fewer consumables during the process.

[0033] For particle bombardment, dried embryonic explants can be plated onto a target culture medium or substrate that can fix the explants in place and be appropriately oriented for blasting. This target culture medium or substrate may contain, for example, gelling agents such as agar and carboxymethyl cellulose (CMC) to control the viscosity of the medium or substrate. Placing the explants in liquids such as hydration, pre-culture, or rinsing media can promote the spreading and positioning of the explants. According to certain embodiments, the explants targeted by particle bombardment can be positioned such that the meristematic tissue of the explant preferentially receives the blasting particles. For example, the explants can be placed on a surface with their meristematic side facing upwards to preferentially receive the coated particles during bombardment. Each explant can also be blasted with coated particles under different pressures, forces, and / or once or multiple times.

[0034] According to embodiments of the present disclosure of co-delivering a selectable marker gene with a DNA sequence encoding one or more genome editing agents, after bombardment or transformation, targeted explants can be cultured on (or in) a post-culture selective medium (or a series of selective media) to allow cells and tissues containing the selectable marker gene to regenerate or develop into plants or plant parts (such as roots and / or shoots) or to select said cells and tissues. The selectable marker gene may be co-delivered with a DNA sequence encoding one or more genome editing agents to select cells that may express one or more genome editing agents and the selectable marker gene. Generally, the selective medium will contain a selector to favor or benefit the survival, growth, proliferation, and / or development of explant cells based on the expression of the selectable marker gene in at least one cell of the explant (when expressed in one or more recipient cells and their progeny cells, the selectable marker gene provides tolerance to the selector). However, according to some embodiments, bombarded or transformed explants may not be subjected to selective pressure, and ultimately, developed or regenerated plants may be screened for the presence of editing or mutations at the target site.

[0035] However, according to some embodiments, explants may optionally be cultured immediately after transformation or bombardment of the target explants on a first post-culture resting medium (or medium) lacking a selector for an initial period of time to allow the explants to recover and / or begin expressing the selection marker gene. The duration of this resting step may range from about 1 hour to about 24 hours, or from about 6 hours to about 18 hours, or from about 10 hours to about 15 hours (e.g., about 12 hours or overnight). While recovery of edited plants can be improved by having a non-selective recovery period (e.g., cultured on resting medium), the frequency of recovery of edited plants may decrease if selection begins too late (e.g., more than 18 to 24 hours after bombardment). Each of the post-culture medium, selection medium, or resting medium may include standard plant tissue culture medium components, such as salts, sugars, plant growth regulators, etc., and may be cultured on these media under standard or different temperature (e.g., 28°C) and light conditions (e.g., 16 / 8 hour photoperiod). However, depending on the editing frequency and selection scheme, such as the specific selection marker genes and selectants used, a first post-culture or resting step may be included or omitted before selection.

[0036] Following any initial recovery and culture of the explants on a first non-selective resting medium, the explants may optionally undergo a enhancement step. According to these embodiments, the explants may be exposed to or placed on (or in) a second post-bombardment or enhancement medium containing an osmotic agent (such as polyethylene glycol (PEG) and / or a calcium salt compound (such as 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%, but their concentrations may vary. This enhancement medium may also lack a selector. Exposing the bombarded explants to the enhancement medium can further drive the entry of coating particles and / or DNA and marker gene constructs encoding one or more genome editing agents (if used) into the explant cells. The explants may be placed in or on the enhancement medium for only a short period, such as from about 30 minutes to about 2 hours, or about 1 hour, and then one or more washing steps may be performed before any further culture or selection steps.

[0037] As mentioned above, bombarding or transforming explants can be contacted with one or more selective media containing a selector to favor the survival, growth, proliferation, and / or development of cells expressing a selector marker gene construct used for co-transformation. The selector marker gene is typically paired with a selector used for selection, thereby conferring tolerance to selection by the selector. For example, the selector marker gene could be an adenylate transferase gene (…). aadA It imparts tolerance to spectinomycin or streptomycin as selectants.

[0038] Plant selection marker genes or transgenes may include any gene that confers tolerance to a corresponding selector, such that plant cells transformed with a plant selection marker transgene are tolerant to and withstand the selection pressure exerted by the selector. Therefore, explant cells receiving the selection marker gene are favored for growth, proliferation, and development under selection. While plant selection marker genes are commonly used to confer tolerance to selectors, one or more other selection markers or reporter genes may also be used. Such selection markers or reporter genes may include, for example, β-glucuronidase (GUS; e.g., as described in, for example, U.S. Patent No. 5,599,670) or green fluorescent protein and its variants (GFP, e.g., as described in U.S. Patent Nos. 5,491,084 and 6,146,826). A variety of selection markers or reporter genes detectable in plants, plant parts, or plant cells are known in the art, such as luciferase, other non-GFP fluorescent proteins, and genes that confer a detectable phenotype in plants, plant parts, or seeds (e.g., phytonene synthase, etc.). Other examples of screening markers may include secretory markers, such as the opine synthase gene, whose expression causes the secretion of one or more molecules that can be detected as a means of identifying transformed cells.

[0039] Plant selection marker genes may include genes encoding proteins that provide or confer tolerance or resistance to herbicides such as glyphosate and glufosinate. Useful plant selection marker genes are known in the art and may include plant selection marker genes encoding proteins that confer resistance or tolerance to: streptomycin or spectinomycin (e.g., adenylate transferase, ...). aadA or spec / strep Kanamycin (e.g., neomycin phosphotransferase or...) nptII Hygromycin B (e.g., hygromycin phosphotransferase, hpt , hp or aph IV ), gentamicin (e.g., aac3 and aacC4 ) and chloramphenicol (e.g., chloramphenicol acetyltransferase or CATOther examples of known plant selection marker genes encoding proteins that confer herbicide resistance or tolerance include, for example, transcribed DNA molecules encoding 5-enolpyruvate shikimate-3-phosphate (EPSP) synthase (EPSP for glyphosate tolerance; e.g., as described in U.S. Patent Nos. 5,627,061, 5,633,435, 6,040,497, and 5,094,945); transcribed DNA molecules encoding glyphosate oxidoreductase and glyphosate-N-acetyltransferase (GOX; e.g., as described in U.S. Patent No. 5,463,175; GAT; U.S. Patent Publication No. 2003 / 0083480); and transcribed DNA molecules encoding phytopene desaturase (…). crtI For example, as Misawa et al., Plants Journal , 4:833-840 (1993); encoding dicamba monooxygenase (DMO) gene (e.g., US application numbers (2003 / 0115626 and 2003 / 0135879); and Behrens et al., Science Transcribed DNA molecules (316(5828):1185 2007) targeting dicamba tolerance; and Misawa et al., Plant Journal (as described in 6:481-489 (1994) regarding tolerance to norflurazon) and resistance to bifenthrin. bar ) or glufosinate N-acetyltransferase ( pat Genes (e.g., those described in DeBlock et al., EMBO Journal, 6:2513-2519 (1987) regarding tolerance to glufosinate and diammonium phosphate).

[0040] To perform one or more selection steps, explants may be contacted with or placed on (or in) one or more selective media containing a selector. In addition to applying selection pressure, the selective media may simultaneously provide the regeneration or development of shoots, roots, and / or the whole plant from the bombarded explants. Alternatively, regeneration media may be used to develop or regenerate one or more shoots and / or roots in the absence of a selector. Regeneration and / or selection media may contain various standard plant tissue culture components, such as salts (e.g., MS or B5 salts), one or more sugars, etc. Regeneration and / or selection media may optionally include one or more plant growth regulators, such as auxins and / or cytokinins, which promote or assist the development, elongation, or regeneration of shoots and / or roots (and ultimately the whole plant). One or more regeneration and / or selection steps may be performed within a range of standard or different temperature (e.g., 28°C) and light conditions (e.g., 16 / 8 photoperiod). This process of developing genome-edited R0 plants from bombarded explants on selective media may largely resemble normal germination and plant development; however, some reorganization of meristematic tissue may occur in response to selective pressure to form buds and / or roots and other plant parts of the adult plant. Importantly, not only is the callus stage prior to the bombardment or transformation step avoided, but the explants can further develop or regenerate genome-edited R0 plants without the formation of embryogenic callus from the explants after transformation.

[0041] According to embodiments of this disclosure, explants can be cultured in a first selective medium (or a series of selective media) until green shoots form, after which they can be removed or cut and transferred to a new selective medium. The transfer or subculture process can be repeated once or several times (e.g., 2, 3, 4, or 5 times) to provide multiple rounds of transfer, subculture, and / or selection. It is believed that multiple rounds of transfer, subculture, and / or selection from shoots of explants under selective pressure can expand or increase the number, proportion, and / or prevalence of genome-edited cells in the entire genome-edited R0 plantlet that develops or regenerates later.

[0042] According to some embodiments, the regeneration medium may also be a selective medium, such as one or more of the selective media described above, and may also be used as a rooting medium to induce or allow the formation and development of one or more roots from one or more buds transferred or subcultured. The rooting medium may contain one or more plant growth regulators, such as auxins and / or cytokinins. The one or more rooting media may also each be a selective medium and, in addition to one or more plant growth regulators, contain a selector. Rooted plantlets developed or regenerated from bombarded explants (through a series of transfers or subcultures under selective pressure) may eventually be transferred to PlantCon. TMOr other suitable containers and / or potting soil are used for the continued development of genome-edited R0 plants, from which genome-edited R1 seeds can then be harvested. Only a few rounds of sequential subculturing (and eventual rooting) of green shoots from the initially bombarded or transformed explants under selection pressure are sufficient to form genome-edited R0 plantlets, which can then further develop into fertile plants producing genome-edited R1 plants and seeds. This disclosure illustrates a significant advance and improvement in the art by providing a means of producing genome-edited plants at reasonable frequencies in diverse plant germplasms. In fact, the method of this disclosure avoids the need for a callus period at any stage of the entire process of preparing dry explants for bombardment or transformation, and then culturing or regenerating genome-edited R0 plants from the bombarded or transformed explants. In contrast, existing methods for genome editing are generally limited to certain explant types and plant germplasms and varieties suitable for a wider range of culture steps.

[0043] According to embodiments of this disclosure, one or more selection steps can be performed in a single selection medium, or more preferably in a series of selection steps or media. The amount or concentration of the selector in the selection medium can vary depending on the specific selector used. For example, for selecting marker genes. aadA The amount of spectinomycin 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 selectant may be kept constant throughout the selection period, or the amount or concentration of the selectant may be gradually increased or increased during the selection period. A stepwise approach allows one or more transformed explant cells to recover for a longer period until they achieve more robust expression of the selection marker gene to withstand stronger selection stress. However, at the initial selection stress, the expression of the selection marker gene may be sufficient, making a stepwise selection approach unnecessary. Regardless of the method, explants may be periodically transferred or subcultured to fresh selection medium, or the selection medium may be periodically replaced and refreshed with new selection medium. According to some embodiments, explants may be held in or on each selection medium for a duration ranging from about a few days (e.g., 2 or 3 days) to several weeks (e.g., 3 to 4 weeks), or from about 1 week to about 3 weeks, or for about 2 weeks, before being transferred or subcultured to the next medium. According to a specific implementation involving the use of spectinomycin as a selector, the concentration of spectinomycin can be increased stepwise from about 50 ppm to about 500 ppm, or alternatively, the concentration of spectinomycin can be kept relatively constant (e.g., about 100 ppm, about 150 ppm, or about 200 ppm).

[0044] According to some embodiments, DNA encoding one or more genome editing agents 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-velocity microjet, microinjection, electroporation, direct DNA uptake, and bacterial-mediated transformation. According to some embodiments, DNA encoding one or more genome editing agents for genome editing can be transformed into at least one cell of a mature embryonic explant via bacterial-mediated transformation. Known bacteria mediating plant cell transformation include those from the Rhizobium family (…). Rhizobiaceae ) and Rhizobium ( Rhizobia Many species in the family ) including, but not limited to, species of the genus *Agrobacterium* and species of the genus *Sinobacterium*. Sinorhizobium sp. ), species of the genus *Mesophytic* (*Rhizobium*) Mesorhizobium sp ) and species of the genus *Slow-growing Rhizobium* ( Bradyrhizobium sp. (See, for example, Broothaerts et al., 2005; and U.S. Patent Application Publications 2007 / 0271627 and 2008 / 0280361).

[0045] DNA molecules can be delivered via bacterial-mediated delivery (e.g., Agrobacterium-mediated delivery; see also, for example, U.S. Patent Nos. 5,563,055, 5,591,616, 5,693,512, 5,824,877, and 5,981,840) to cells in the living meristem of an embryo excised from a seed (such as soybean and other crop seeds). The meristem region can be cultured in the presence of a selector to regenerate one or more RO plants transformed with DNA molecules. Shoot and / or root formation can occur in a variety of culture media to regenerate plants from one or more embryonic explant cells, which may include transformed meristem cells.

[0046] Possible selectors may include auxin-like herbicides such as dicamba or 2,4-D, MCPA, glufosinate, acetolactate synthase inhibitors, protoporphyrinogen oxidase inhibitors and hydroxyphenyl-pyruvate-dioxygenase inhibitors, neomycin, kanamycin, paramomycin, G418, aminoglycosides, spectinomycin, streptomycin, hygromycin B, bleomycin, phleomycin, sulfonamides, streptomycin, chloramphenicol, methotrexate, 2-deoxyglucose, betaine, S-aminoethyl-L-cysteine, 4-methyltryptophan, D-xylose, D-mannose, and benzyladenine-N-3-glucuronidase. Examples of selective marker genes for resistance to these selectors are also known in the art.

[0047] Various tissue culture media are known that, when appropriately supplemented, support plant tissue growth and development, including the formation of mature plants from excised meristems or embryos. These tissue culture media can be purchased as commercial preparations or custom-prepared and 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 derivative media supplemented with these media. Those skilled in the art will understand that media and supplementary media (such as nutrients and growth regulators) used for transformation and regeneration are typically optimized for a specific target crop or variety of interest. The reagents are commercially available and can be purchased from many suppliers (see, for example, Sigma Chemical Co., St. Louis, Mo. and Phytotechnology Laboratories, Shawnee Mission, Kans.).

[0048] Co-culture and subsequent steps can be performed in darkness or in a lit Percival incubator for, for example, 2 to 5 days, with a photocycle of 16 hours of light followed by 8 hours of darkness. In one embodiment, the light intensity may be, for example, at least about 5 uE, including at least about 10 uE or 25 uE, including between about 5 uE and about 200 uE, or other light conditions that allow normal plastid development at about 23 to 25°C and can be performed at up to about 35°C.

[0049] This disclosed method allows for the regeneration and / or development of candidate genome-edited plants from one or more bombarded or transformed explants without extensive culture, thus increasing the efficiency of identifying and growing shoots and plants containing one or more genome-edited cells and reducing the cost and labor required to produce genome-edited plants of desired varieties or germplasm. For example, after identifying the putative transformant using selection markers, the plantlets can be placed in soil or on soil substitutes, such as rooting media, with or without a selection agent. Molecular techniques can be used to analyze whether genome editing is present at the target site in the shoots elongating from the selected or regenerated explants. Genome-edited R0 plants can further produce genome-edited R1 plants and seeds, which can produce subsequent progeny plants and seeds that are also genome-edited. While genome-edited R0 plants can be produced by this disclosed method with minimal selection pressure, selection can be maintained with an appropriate selection agent throughout one or more culture or regeneration steps. R1 plants with one or more genome edits at the desired target site are identified as being hybridizable to another plant, and homozygous genome-edited plants can be selected from the next generation of plants with one or more genome edits or mutations that are genetically fixed relative to the offspring (in the case of one or more edits or mutations in non-segregating offspring plants and stable homozygosity in self-pollinated offspring). As described above, the growth, survival, development, etc., of genome-edited cells in R0 plants can also be selectively or preferentially achieved or promoted by applying selection pressure with a selecting agent during one or more culture, subculture, shoot elongation, and / or rooting steps of the explant, to produce R0 plants with a large proportion of cells with one or more genome edits or mutations due to the co-delivery of the selection marker gene, but the selection pressure (e.g., in the form of local spraying, soil or seed application, etc.) can be continued alternately (e.g., periodically, etc.) after the initial culture and / or during the remaining lifespan of the R0 plant.

[0050] Various tissue culture media are known that, when appropriately supplemented, support plant tissue growth and development, including the formation of mature plants from excised plant tissue. These tissue culture media are available as commercial formulations or can be custom-prepared and modified by those skilled in the art. Examples of such media include, but are not limited to, those described below: Murashige and Skoog. Physiol. Plant 15:473-497, 1962); Chu et al., ( Chinese Science 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., Plant 165:322-332, 1985; McCown and Lloyd, HortScience 16:453, 1981; Nitsch and Nitsch Plant Physiol. 44:1747-1748, 1969; and Schenk and Hildebrandt, Canon. J. Bot. 50:199-204, 1972; or derivatives thereof supplemented accordingly. Those skilled in the art will recognize that tissue media optimized for transformation, selection, and regeneration, typically for a specific target crop or variety of interest, may be supplemented with carbohydrates, such as, but not limited to, glucose, sucrose, maltose, mannose, fructose, lactose, galactose, and / or glucose, or carbohydrates in various proportions. Reagents are commercially available and from numerous suppliers (see, e.g., Sigma Chemical Co., St. Louis, MO; and PhytoTechnology Laboratories, Shawnee Mission, KS). These tissue media may be used as resting media, as selection media further supplemented with selectants, and / or as regeneration media when supplemented with one or more plant growth regulators.

[0051] Embodiments of this disclosure also provide genome-edited plants, plant parts, and seeds generated by the transformation methods of this disclosure, which contain 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 some embodiments of this disclosure, the plant or plant part is a seed.

[0052] II. Transformable explants The method disclosed herein may also include one or more steps of removing at least a portion of the plant embryo from the plant seed by any suitable manual or automated method prior to transformation. According to embodiments of this disclosure, suitable embryo explants further comprise the meristem / meristematic tissue of the embryo, or at least a portion of the meristem, or at least one meristematic cell of the embryo explant, because targeting the meristematic cell of the explant for transformation to achieve delivery of DNA encoding one or more genome editing agents can improve the efficient production and development or regeneration of genome-edited plants or is necessary for it. Embryo explants may lack one or more embryogenic tissues, such as one or more cotyledons, one or more hypocotyls, radicles, etc., as long as they retain at least a portion of the embryonic meristem. According to many embodiments of this disclosure, the use of mature embryo explants removed from dry seeds may be preferred, however, they may require one or more hydration and / or pre-culture steps prior to transformation.

[0053] Any suitable method for producing or removing embryonic explants from plant seeds may be used in conjunction with embodiments of this disclosure. These methods may be automated and / or manual, and may involve singulated or bulk processes. According to many embodiments, the embryonic explant may be a mature embryonic explant (or a portion thereof) obtained or removed from a dried mature plant seed. For any given plant species, a mature seed or embryo may be defined with respect to a number of post-pollination days (DAP) greater than or equal to a certain number to distinguish immature seeds or embryos of the same plant species, but the transition from immature to mature embryo in a given plant species 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 (among other developmental changes).

[0054] Since the development or maturation of seeds and embryos is accompanied by drying, the mature seeds or embryo explants used in the methods of this disclosure can also be defined with respect to their moisture content. Furthermore, embryo explants can be defined based on the moisture content of the seeds from which they are excised. For example, the moisture content of the seeds or embryo explants used in the methods according to the invention may initially be the following or within the following ranges: about 3% to about 25%, or about 4% to about 25%, or about 3% or 4% to about 20%, or any percentage value or range within or within such a broader percentage range (depending on the specific plant species), such as about 5% to about 20%, about 5% to about 15%, about 8% to about 15%, and about 8% to about 13%. In practice, plant seeds may be artificially dried or dehydrated before excising the embryo explants, provided that the seeds and embryos remain viable and can be used for plant transformation and development or regeneration, prior to use in embodiments of the methods of this disclosure. Drying seeds can facilitate the excising of embryo explants from seeds and / or the storage of embryo explants derived from seeds. Alternatively or additionally, seeds may be hydrated or swelled prior to explant removal to promote embryonic development, soften the embryo, reduce damage to the embryo, and / or maintain embryonic viability during the explant removal procedure. However, hydration of seeds or explants can reduce or eliminate their storability, even if the seeds or explants are subsequently dried or dehydrated.

[0055] For a further description of embryonic explants and methods for removing embryonic explants from potentially pre-hydrated, induced, or germinated dry, dried, and / or mature seeds, see, for example, U.S. Patent Nos. 8,466,345, 8,362,317, and 8,044,260 and U.S. Patent Publication No. 2016 / 0264983. Regardless of the seed type used and the precise method used for mechanically removing embryonic explants from the seeds, additional steps and processes, such as sterilization and culling, may be performed to prepare and / or enrich explants for particle bombardment. The dry or induced embryonic explants may also be hydrated, induced, and / or germinated after removal but before the transformation step.

[0056] Embryonic explants used with this disclosure can be removed from seeds less than one day before use in the methods of this invention, such as about 1 to 24 hours before use, including about 2, 6, 12, 18, or 22 hours before use. However, according to other embodiments, seeds and / or explants can be stored for longer periods before their use, including days, weeks, months, or even years, depending on the storage conditions used to maintain the viability of the seeds and / or explants. An advantage and benefit of using dried mature seeds as a source for producing or excising embryonic explants suitable for genome editing is that dried mature seeds and / or explants can be stored under dry conditions (not germinating during storage and remaining viable and capable of transformation). Such dry storage conditions can be defined as storage in an environment or surrounding environment with sufficiently low moisture content or humidity such that the stored seeds and / or explants do not germinate and remain viable and capable of transformation for a desired long period before use in the current transformation methods, such as about 1 hour to about 2 years, or about 24 hours to about 1 year, or any specific time period or range of time within those broader time ranges. By using storable seeds or explants, a reliable supply of seed or explant source material can be obtained without the need for donor plants. The ability to store mature dry seeds involves the natural characteristics of dry mature seeds and embryos. In other words, dry mature seeds and / or embryo explants can also be defined with respect to their quiescent, arrested, or low-metabolic state or activity. Therefore, the dry seeds or explants used according to the methods of this disclosure can be defined with respect to their low-metabolic state and / or by their metabolic or developmental quiescent or arrested state until later hydration and germination of the seed or embryo.

[0057] According to some implementations, hydration or germination of the embryonic explant or seed can be performed before or after excision of the embryonic explant from the seed. In other words, in addition to any pre-culture step, the seed can be imbibed or hydrated before excision of the embryonic explant to allow the seed to begin germination and / or development, or alternatively, the dry embryonic explant can be excised from the seed and then imbibed or hydrated to trigger germination and / or development of the embryonic explant. The induced or germinated seed can then be bombarded with particles without prior greening of the target tissue, which can be controlled by the amount of time prior to the transformation step and / or limited light exposure. However, as mentioned above, the hydration step is alternatively used only to hydrate the dry embryonic explant to make the "wet" explant more susceptible to particle bombardment and delivery of DNA encoding one or more genome-encoding reagents without allowing the embryo to germinate or develop further (e.g., the time for the hydration or imbibed step can be limited so that no significant developmental changes and / or germination occur in the embryonic explant before bombardment).

[0058] Explants used in conjunction with the method implementations provided herein may include explants from a variety of monocotyledonous / monocot and dicotyledonous / dicot plants, including agricultural crop species such as maize, wheat, rice, sorghum, oats, barley, sugarcane, African oil palm, switchgrass, cotton, rapeseed, sugar beet, alfalfa, soybean, and other leguminous plants.

[0059] III. Genome Editing The cells, plants, plant parts, and seeds disclosed herein are produced through genomic modifications using site-specific integration or genome editing. Targeted modification of the plant genome via genome editing can be used to produce crop plants with improved traits. Genome editing can be used to produce one or more edits or mutations at desired target sites in the plant genome, such as to alter the expression and / or activity of one or more genes, or to integrate insert sequences or transgenes at desired locations in the plant genome. As used herein, “site-specific integration” refers to genome editing methods and techniques that target the integration or insertion of polynucleotides (e.g., insert sequences, regulatory elements, or transgenes) into the plant genome. As provided herein, DNA molecules encoding one or more genome editing agents can be delivered to recipient cells of explants, such as meristematic cells of explants. Guiding nucleic acids and / or DNA encoding one or more genome editing agents can be delivered to recipient cells of explants via transformation methods without integrating or incorporating polynucleotides or transgenes into the recipient cell genome. The DNA molecule encoding one or more genome editing agents may further include: (i) a transcribed DNA sequence encoding a guide nucleic acid for a guide nuclease; (ii) a marker gene or transgene, such as a selection or screening marker gene; and / or (iii) a donor template.

[0060] According to many embodiments, recombinant DNA constructs or molecules are provided that comprise a sequence encoding one or more genome editing agents, wherein said sequence is operatively linked to a promoter. The promoter may be heterologous with respect to a sequence encoding a site-specific nuclease. The promoter may be a plant-expressible promoter, such as a constitutive promoter, a tissue-specific or tissue-preferred 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 transcribed DNA sequence encoding a guiding nucleic acid, wherein the second sequence is operatively linked to a second promoter; and / or (ii) a marker gene or transgene, which may be a selection or screening marker gene. The second promoter may be heterologous with respect to a sequence encoding a guiding nucleic acid. The second promoter may be a plant-expressible promoter, such as a constitutive promoter, a tissue-specific or tissue-preferred promoter, a developmental stage promoter, or an inducible promoter. The marker gene or transgene may comprise a coding sequence operatively linked to a promoter (such as a heterologous and / or plant-expressible promoter). According to some embodiments, the recombinant DNA construct or molecule may further comprise a donor template region. The recombinant DNA construct disclosed herein can be delivered to recipient explant cells via any transformation method known in the art, such as particle bombardment or bacterial-mediated transformation.

[0061] According to many embodiments, a recombinant DNA construct or molecule comprising a sequence encoding one or more genome editing agents ( wherein the sequence is operatively linked to a promoter) is applied or coated onto particles or beads for use in biological projectile delivery or incorporation into a transformation vector for transformation into explants or explant cells or tissues. The promoter may be heterologous with respect to the genome-encoding agent. The promoter may be a plant-expressible promoter, such as a constitutive promoter, a tissue-specific or tissue-preferred 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 transcribed DNA sequence encoding a guiding nucleic acid, wherein the second sequence is operatively linked to a second promoter. The second promoter may be heterologous with respect to the sequence encoding the guiding nucleic acid. The second promoter may be a plant-expressible promoter, such as a constitutive promoter, a tissue-specific or tissue-preferred promoter, a developmental stage promoter, or an inducible promoter. According to some implementations, a recombinant DNA construct or molecule containing a sequence encoding one or more genome editing agents ( wherein the sequence is operatively linked to a promoter) and one or more of a guide nucleic acid, a marker gene and / or a donor DNA template are applied or coated onto particles or beads for delivery to explants or explant cells or tissues via biological projectiles.

[0062] According to the methods provided herein, DNA constructs or molecules encoding any suitable genome editing reagent (such as zinc finger nucleases (ZFNs), guide nucleases, TALE endonucleases (TALENs), broad-spectrum nucleases, recombinases, transposases, or any combination thereof) can be delivered to explant cells to induce genome editing or site-directed integration at a target site within the genome of the explant cells and / or their progeny cells. In the case of guide nucleic acids (such as clustered regularly interspersed short palindromic repeat (CRISPR) enzymes), the DNA construct or molecule can be co-delivered with the guide nucleic acid to guide the guide nuclease to the target site. Guide nucleases may also include any known homologs or modified forms of guide nucleases that share conserved amino acids and have a high percentage of identity with respect to their respective protein sequences (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity within the alignment length of their protein sequences).

[0063] According to some embodiments, a DNA construct or molecule encoding a genome editing agent may be co-delivered with a donor template molecule to serve as a template for desired editing, mutation, or insertion into the genome at a desired target site by repairing double-strand breaks (DSBs) or gaps generated by the genome editing agent. According to some embodiments, the DNA construct or molecule encoding a genome editing agent may be co-delivered with a DNA molecule containing a selection or screening marker gene. In each case, optionally, in addition to one or more of the guiding nucleic acid, the donor template molecule, and / or the DNA molecule encoding the selection or screening marker, the DNA construct encoding the genome editing agent may be applied to or coated onto a particle for delivery to recipient cells of the explant. According to some implementation schemes, a DNA construct or molecule encoding a genome editing agent may be applied to or coated onto particles used for biological projectile or particle delivery or incorporated into a vector for transformation into one or more recipient cells of an explant, wherein the one or more recipient cells of the explant contain one or more DNA molecules and / or transgenes prior to particle bombardment or transformation, which can be stably transformed into the genome of the recipient cells, wherein such one or more DNA molecules and / or transgenes contain or encode one or more of the following: (i) a donor template molecule to serve as a template for performing desired editing, mutation, or insertion into the genome at a desired target site; (ii) a selection or screening marker gene; and / or (iii) a directing nucleic acid to guide a nuclease to the desired target site.

[0064] Genome editing reagents can be guide nucleases, which act as ribonucleoprotein (RNP) complexes with guide RNA. According to some implementation schemes, the guide nucleases can be selected from: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, and Cas9. (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, 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, Cpf1, CasX, CasY, CasZ and their homologs or modified forms, Argonaute (non-limiting examples of Argonaute proteins include thermophilic bacteria) Thermus thermophilus Argonaute (TtAgo), Thermococcus fibrillosa ( Pyrococcus furious Argonaute (PfAgo), Halophilic bacillus griseus ( Natronobacterium gregoryi Argonaute (NgAgo) and its homologs or modified forms). Depending on some implementations, the guiding nuclease is a Cas9 or Cpf1 enzyme. The DNA construct or molecule encoding the guiding nuclease may or may not be delivered with the guiding nucleic acid.

[0065] For guide nucleases, guide nucleic acid molecules can also be provided to direct the guide nuclease to a target site in the plant genome via base pairing or hybridization, thereby generating a DSB or nick at or near the target site. Guide nucleic acids can be used as guide nucleic acid molecules, or as recombinant DNA molecules, constructs, or vectors containing a transcribed DNA sequence encoding the guide nucleic acid that is operatively linked to a promoter or a plant-expressible promoter, to be transformed or introduced into plant cells or tissues. Promoters can be constitutive promoters, tissue-specific or tissue-preferred promoters, developmental stage promoters, or inducible promoters.

[0066] As used herein, the term "guide nucleic acid" refers to a nucleic acid comprising: a first fragment containing a nucleotide sequence complementary to a sequence in the target nucleic acid; and a second fragment interacting with a guide nuclease protein. In some embodiments, the first guide fragment containing a nucleotide sequence complementary to a sequence in the target nucleic acid corresponds to CRISPR RNA (crRNA or crRNA repeat sequence). In some embodiments, the second guide fragment containing a nucleic acid sequence interacting with a 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 a sequence in the target nucleic acid and a polynucleotide interacting with a guide nuclease protein), and is referred to herein as a "double-guide" or "two-molecule guide." In some embodiments, the double 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 to herein as a "single-molecule guide" or "single guide." In some embodiments, the single guide may comprise DNA, RNA, or a combination of DNA and RNA. The term "guide nucleic acid" is inclusive, referring to both two-molecule and single-molecule guides.

[0067] As is known in the art, a protospacer-adjacent motif (PAM) can be present in the genome immediately adjacent to and upstream of a genomic target site sequence that is complementary to the target sequence of a guide RNA, the target sequence being immediately downstream (3') of the sense (+) strand of the genomic target site (relative to the target sequence of the guide RNA). 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 (relative to the target sequence of the guide RNA) may contain 5'-NGG-3'. However, the corresponding sequence of the guide nucleic acid (immediately downstream (3') of the target sequence of the guide RNA) may not typically be complementary to the genomic PAM sequence.

[0068] The guide nucleic acid is typically a non-coding RNA molecule that does not encode a protein. The target sequence of the guide nucleic acid can be at least 10 nucleotides long, such as 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, or about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides long. The target sequence may have at least 95%, at least 96%, at least 97%, at least 99%, or 100% identity or complementarity with 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 genomic target site.

[0069] In addition to the target sequence, the guiding nucleic acid may also contain one or more other structural or scaffold sequences that can bind to or interact with RNA-guided nucleases. Such scaffold or structural sequences may also interact with other RNA molecules (e.g., tracrRNA). Methods and techniques for designing targeting constructs and guiding nucleic acids for genome editing and site-specific integration using guiding nucleases at target sites within the plant genome are known in the art.

[0070] Several site-specific nucleases (such as recombinases, zinc finger nucleases (ZFNs), broad-spectrum nucleases, and TALENs) are not nucleic acid-guided; instead, they rely on their protein structure to determine the target sites for creating DSBs or nicks, or they are fused, tethered, or linked to DNA-binding protein domains or motifs. The protein structure of site-specific nucleases (or fused / linked / tethered DNA-binding domains) allows them to target specific sites. According to many of these embodiments, non-nucleic acid-guided site-specific nucleases (such as recombinases, zinc finger nucleases (ZFNs), broad-spectrum nucleases, and TALENs) can be designed, engineered, and constructed using known methods to target and bind to genomic loci of endogenous genes in plants to generate DSBs or nicks at such loci, thereby knocking out or reducing gene expression through DSB or nick repair. This can lead to sequence mutations or insertions at the DSB or nick site via cellular repair mechanisms that can be directed by donor template molecules.

[0071] In some embodiments, the site-specific nuclease is a recombinase. The recombinase may be a serine recombinase linked to a DNA recognition motif, a tyrosine recombinase linked to a DNA recognition motif, or other recombinases known in the art. The recombinase or transposase may be a DNA transposase or recombinase linked to or fused to a DNA-binding domain. Non-limiting examples of recombinases include tyrosine recombinases linked to the DNA recognition motifs provided herein, selected from Cre recombinases, etc. Gin A group consisting of recombinases, Flp recombinases, and Tnp1 recombinases. In one aspect, the Cre recombinase or... provided herein... Gin The recombinase is tethered to a zinc finger DNA-binding domain, or a TALE DNA-binding domain, or a Cas9 nuclease. Alternatively, the serine recombinase ligated to the DNA recognition motif provided herein is selected from the group consisting of PhiC31 integrase, R4 integrase, and TP-901 integrase. Alternatively, the DNA transposase ligated to the DNA-binding domain provided herein is selected from the group consisting of TALE-piggyBac and TALE-Mutator.

[0072] Site-specific nucleases can be zinc finger nucleases (ZFNs). ZFNs are synthetic proteins composed of engineered zinc finger DNA-binding domains fused to a cleavage domain (or cleavage hemidomain), and can be derived from restriction endonucleases (e.g., FokI The DNA-binding domain can be typical (C2H2) or atypical (e.g., C3H or C4). Depending on the target site, the DNA-binding domain may contain one or more zinc fingers (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more zinc fingers). Multiple zinc fingers in the DNA-binding domain can be separated by one or more adapter sequences. ZFNs can be designed to cleave almost any segment of double-stranded DNA through modification of the zinc finger DNA-binding domain. ZFNs consist of non-specific DNA cleavage domains fused with the DNA-binding domain (e.g., derived from...). FokI The monomers of a nuclease form a dimer, and the DNA-binding domain includes an array of zinc fingers engineered to bind to target site DNA sequences. The DNA-binding domain of a ZFN typically consists of 3 to 4 (or more) zinc fingers. The amino acids at positions -1, +2, +3, and +6 relative to the origin of the α-helix of the zinc fingers that facilitates site-specific binding to the target site can be altered and customized to fit a specific target sequence. Other amino acids can form a common backbone to produce ZFNs with different sequence specificities.

[0073] Methods and rules for designing ZFNs that target and bind to specific target sequences are known in the art. See, for example, U.S. Patent Applications Nos. 2005 / 0064474, 2009 / 0117617, and 2012 / 0142062. FokI Nuclease domains may require dimerization to cleave DNA and therefore require two ZFNs with their C-terminal regions to bind to opposite strands of DNA (5-7 bp apart) at the cleavage site. If the dual ZF binding sites are palindromic, then the ZFN monomer can cleave the target site. As used herein, ZFN is broad and includes monomeric ZFNs that can cleave double-stranded DNA without the assistance of another ZFN. The term ZFN can also be used to refer to one or both members of a pair of ZFNs engineered to work together to cleave DNA at the same site. Without being limited by any theory, because the DNA-binding specificity of zinc finger domains can be reengineered using one of a variety of methods, custom-designed ZFNs can theoretically be constructed to target virtually any target sequence (e.g., at or near a gene in a plant genome). Publicly available methods for engineering zinc finger domains include context-dependent assembly (CoDA), oligomerized pool engineering (OPEN), and modular assembly. In one aspect, 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 aspect, the ZFNs provided herein are capable of producing targeted DSBs or incisions.

[0074] Site-specific nucleases can be TALENs. TALENs are formed by combining a transcription activator-like effector (TALE) DNA-binding domain with a nuclease domain (e.g., ...). FokI Artificial restriction enzymes are produced through fusion. When each member of a TALEN pair binds to a DNA site flanking the target site, FokI Monomerization occurs, causing double-strand DNA breaks at the target site. (Except for wild-type) FokI Beyond the cleavage domain, structures with mutations have been designed. FokI Variations of the cleavage domain are used to improve cleavage specificity and cleavage activity. FokI The domain functions as a dimer, requiring two constructs with unique DNA-binding domains for sites in the target genome with appropriate orientation and spacing. The TALEN DNA-binding domain and... FokI The number of amino acid residues between cleavage domains and the number of bases between two individual TALEN binding sites are parameters for achieving high activity levels.

[0075] TALENs are artificial restriction enzymes created by fusing a transcription activator-like effector (TALE) DNA-binding domain with a nuclease domain. In some respects, nucleases are selected from the group consisting of: PvuII , MutH , TV , FokI、AlwI、MlyI、SbfI、SdaI、StsI、CleDORF、Clo051 and Pept071 When each member of the TALEN pair binds to a DNA site flanking the target site, FokI Monomerization occurs, resulting in double-stranded DNA breaks at the target site. As used herein, the term TALEN is broad and includes monomeric TALENs that can cleave 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 cleave DNA at the same site.

[0076] Transcription activator-like effectors (TALEs) can be engineered to bind virtually any DNA sequence, such as those at or near genomic loci in plants. A TALE has a central DNA-binding domain consisting of 13 to 28 repeating monomers of 33 to 34 amino acids each. Except for the highly variable amino acid residues at positions 12 and 13, the amino acids in each monomer are highly conserved. Two variable amino acids are called repeat variable diresidues (RVDs). The amino acids in RVDs preferentially recognize adenine, thymine, cytosine, and guanine / adenine for NI, NG, HD, and NN, respectively, and regulation of RVDs can recognize consecutive DNA bases. This simple relationship between amino acid sequence and DNA recognition allows for the engineering of specific DNA-binding domains by selecting combinations of repeating sequence segments containing appropriate RVDs.

[0077] Except for wild type FokI Beyond the cleavage domain, structures with mutations have been designed. FokI Variations of the cleavage domain are used to improve cleavage specificity and cleavage activity. FokI The domain functions as a dimer, requiring two constructs with unique DNA-binding domains for sites in the target genome with appropriate orientation and spacing. The TALEN DNA-binding domain and... FokI The number of amino acid residues between cleavage domains and the number of bases between two individual TALEN binding sites are parameters for achieving high activity levels. PvuII, MutH and TV The cut structure domain is intended for use with TALE. FokI and FokI Available alternatives to the variant. When coupled with TALE, PvuII It functions as a highly specific cleavage domain (see Yank et al. 2013). PLoS One . 8: e82539). MutH It can introduce strand-specific nicks into DNA (see Gabsalilow et al., 2013). Nucleic Acids Research . 41: e83). TV Introducing double-strand breaks in DNA at the target site (see Beurdeley et al., 2013). Nature Communications . 4: 1762).

[0078] The relationship between amino acid sequences and DNA recognition of TALE-binding domains allows for the designability of proteins. Software programs such as DNAWorks can be used to design TALE constructs. 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 respect, the TALEN provided in this paper is capable of generating targeted DSBs.

[0079] Site-specific nucleases can be broad-spectrum nucleases. Broad-spectrum nucleases, typically identified in microorganisms, such as the LAGLIDADG family of homing endonucleases, are unique enzymes with highly active, site-specific recognition sequences (>14 bp) that induce site-specific digestion of target DNA. Engineered forms of naturally occurring broad-spectrum nucleases often possess extended DNA recognition sequences (e.g., 14 to 40 bp). According to some embodiments, broad-spectrum nucleases may contain selected... CreI , I-CeuI , I-MsoI , I-SceI , I-AniI and DmoI The scaffold or base enzyme that makes up the group. Engineering large-scale nucleases can be more challenging than ZFNs and TALENs because their DNA recognition and cleavage functions are intertwined within a single domain. Specialized mutagenesis and high-throughput screening methods have been used to generate novel large-scale nuclease variants that recognize unique sequences and possess modified nuclease activity. Therefore, large-scale nucleases can be selected or engineered to bind to genomic target sequences in plants, such as those at or near genomic loci of genes. In another respect, the large-scale nucleases presented herein are capable of generating targeted DSBs.

[0080] According to some implementation schemes, a donor template may be co-delivered with a DNA construct or molecule encoding a genome editing agent to the recipient cells of an explant to serve as a template for generating the desired edit during the repair of double-strand breaks (DSBs) or nicks at target sites in the recipient cell genome by the genome editing agent. Alternatively, the donor template may already be present in the recipient cells of the explant. Similarly, for guide nucleases, a transcribed DNA sequence or a transgene encoding a guide nucleic acid may be co-delivered with a DNA construct or molecule encoding a guide nuclease to the recipient cells of an explant to serve as a guide for the guide nuclease to form double-strand breaks (DSBs) or nicks at desired loci or target sites in the recipient cell genome. Alternatively, the guide nucleic acid and / or a DNA molecule or transgene containing a transcribed DNA sequence encoding the guide nucleic acid may already be present and / or expressed by the recipient cells of the explant.

[0081] According to some implementation schemes, (i) a DNA construct or molecule encoding a genome editing reagent, a guide nucleic acid, and a donor template may be applied to or coated onto a particle for delivery of a biological projectile to a recipient cell; or (ii) a DNA construct or molecule encoding a genome editing reagent and / or a guide RNA may be applied to or coated onto a particle for delivery of a biological projectile to a recipient cell, wherein the donor template may optionally be present in or expressed in the recipient cell; or (iii) a DNA construct or molecule encoding a genome editing reagent and / or a donor template may be applied to or coated onto a particle for delivery of a biological projectile to a recipient cell, wherein the guide nucleic acid may optionally be present in or expressed in the recipient cell. Optionally present in or expressed in recipient cells; or (iv) a guiding nucleic acid and / or donor template may be applied to or coated onto the particle for delivery of a biological projectile to the recipient cell, and the genome editing reagent or a DNA construct or molecule encoding the genome editing reagent may optionally be present in or expressed in the recipient cell; in each case, (i), (ii), (iii) or (iv) a double-strand break (DSB) or nick is formed at a desired locus or target site in the recipient cell genome by means of the genome editing reagent, thereby producing templated or non-templated editing or mutation at a desired location in the recipient plant cell genome.

[0082] Any site or locus within the plant genome can potentially be selected for genome editing (or gene editing) or site-specific integration of transgenic, construct, or transcribed DNA sequences. For genome editing and site-specific integration, a double-strand break (DSB) or nick can first be formed at the selected genomic locus equipped with genome editing reagents such as zinc finger nucleases (ZFNs), engineered or naturally occurring broad-spectrum nucleases, TALE endonucleases, or guide endonucleases (e.g., Cas9 or Cpf1). Any method known in the art for site-specific integration can be used. In the presence of a donor template molecule with the insert sequence, the DSB or nick can be repaired via homologous recombination between one or more homologous arms of the donor template and the plant genome, or via non-homologous end joining (NHEJ), resulting in site-specific integration of the insert sequence into the plant genome to produce a targeted insertion event at the DSB or nick site. Therefore, if the transgene, transcribed DNA sequence, construct, or sequence is located in the insertion sequence of the donor template, site-specific insertion or integration of the transgene, transcribed DNA sequence, construct, or sequence can be achieved.

[0083] The introduction of DSBs or notches can also be used to introduce targeted mutations in plant genomes. According to this method, mutations such as deletions, insertions, inversions, and / or substitutions can be introduced at target sites via incomplete repair of DSBs or notches to produce gene knockouts or subtractions. Such mutations can be generated even without the use of donor template molecules via imperfect repair of targeted loci. Gene “knockout” can be achieved by inducing DSBs or notches at or near endogenous loci of a gene, resulting in the non-expression of a protein or the expression of a non-functional protein; similarly, gene “subtraction” can be achieved by inducing DSBs or notches at or near endogenous loci of a gene, which are incompletely repaired at sites that do not affect the coding sequence of the gene in a manner that would eliminate the function of the edited protein. For example, the site of a DSB or notch within an endogenous locus can be located upstream of the gene or in the 5' region (e.g., promoter and / or enhancer sequences) to affect or reduce its expression level. Similarly, such targeted knockout or knockdown mutations in genes can be generated using donor template molecules to guide 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 relative to the target genomic sequence at or near the DSB or nick site, with or without an insertion sequence and containing one or more mutations, such as one or more deletions, insertions, inversions, and / or substitutions. For example, a targeted knockout mutation in a gene can be achieved by substituting, inserting, deleting, or inverting at least a portion of the gene, such as by introducing a frameshift or early stop codon into the coding sequence of the gene. The deletion of a portion of a gene can also be introduced by generating DSBs or nicks at two target sites and causing a deletion of an intercalary target region flanking the target site.

[0084] As used herein, a “donor molecule” may be a recombinant polynucleotide, DNA, or RNA donor template or sequence. A “donor template” or “donor template molecule” (collectively, “donor template”) is defined as a nucleic acid molecule having a homologous nucleic acid template or sequence (e.g., a homologous sequence) and / or an insert sequence for site-directed, targeted insertion or recombination into the plant cell genome via repair of nicks or double-strand DNA breaks in the plant cell genome. A donor template may be a single DNA molecule containing one or more homologous sequences and / or an insert sequence for targeted integration, or a sequence portion (e.g., a donor template region) of a DNA molecule that also contains one or more other expression cassettes, genes / transgenic structures, and / or transcribed DNA sequences. For example, a “donor template” may be used for site-directed integration of transgenic or repressor constructs, or as a template for introducing mutations such as insertions, deletions, or substitutions into target sites within the plant genome. The targeted genome editing techniques described herein may include the use of one or more, two or more, three or more, four or more, or five or more donor molecules or templates. The "donor template" can be a single-stranded or double-stranded DNA or RNA molecule or plasmid. The "insert sequence" of the donor template is a sequence designed for targeted insertion into the plant cell genome, and it can have any suitable length. For example, the length of the insert sequence of the donor template can be between 2 and 50,000, 2 and 10,000, 2 and 5,000, 2 and 1,000, 2 and 500, 2 and 250, 2 and 100, 2 and 50, 2 and 30, 15 and 50, 15 and 100, 15 and 500, 15 and 1,000, 15 and 5,000, 18 and 30, 18 and 26, 20 and 26, 20 and 50, 20 and 100, 20 and 250, 20 and ... Nucleotides or base pairs between 500, 20 and 1000, 20 and 5000, 20 and 10000, 50 and 250, 50 and 500, 50 and 1000, 50 and 5000, 50 and 10000, 100 and 250, 100 and 500, 100 and 1000, 100 and 5000, 100 and 10000, 250 and 500, 250 and 1000, 250 and 5000, or 250 and 10000. The donor template may also have at least one homologous sequence or homologous arm, such as two homologous arms, to integrate the mutated or inserted sequence into the target site within the plant genome via homologous recombination, wherein the homologous sequence or said one or more homologous arms are identical or complementary to the sequence at or near the target site within the plant genome, or have a certain percentage of identity or complementarity.When the donor template includes one or more homologous arms and an insertion sequence, the one or more homologous arms will be side-attached to or around the insertion sequence of the donor template.

[0085] According to some embodiments, the donor template may include a "donor template region" of a recombinant polynucleotide molecule or construct that serves as a donor template for site-specific integration of an insert sequence or template-mediated repair, wherein the recombinant polynucleotide molecule or construct also includes other elements independent of the donor template region. For example, the recombinant polynucleotide molecule or construct may include a "donor template region" and one or more transgenes, such as selectable markers and / or transcribed DNA sequences encoding non-coding RNA molecules (such as guide RNA or RNA molecules for repressing target genes).

[0086] The insert sequence of the donor template may contain one or more genes or sequences, each encoding a transcribed non-coding RNA or mRNA sequence and / or a translated protein sequence. The transcribed sequence or gene of the donor template may encode a protein or non-coding RNA molecule. The non-coding RNA molecule may be, for example, a guide RNA or an RNA molecule targeting a repressor gene (e.g., microRNA (miRNA), small interfering RNA (siRNA), antisense RNA strands, inverted repeat sequences, etc.). The insert sequence of the donor template may contain a polynucleotide sequence that does not contain a functional gene or a complete gene sequence (e.g., the donor template may contain only regulatory sequences, such as promoter sequences, or only a portion of a gene or coding sequence), or may not contain any gene sequence that can identify gene expression elements or any actively transcribed gene sequence. Furthermore, the donor template may be linear or circular, and may be single-stranded or double-stranded. The donor template may be delivered to the cell in the form of a DNA molecule or an RNA molecule expressed from a transgene. Donor templates can be delivered to cells in the form of naked nucleic acid molecules or in the form of complexes with one or more delivery agents (e.g., liposomes, proteins, poloxamer, protein-encapsulated T-chains, etc.). The insert sequence of the donor template provided herein may comprise 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 operatively linked to a promoter and / or other regulatory sequences, such as constitutive, inducible, or tissue-specific promoters.

[0087] According to some embodiments, the donor template may not contain an insert sequence, but instead contains one or more homologous sequences, said homologous sequences including one or more mutations, such as insertions, deletions, substitutions, etc., relative to a genomic sequence at a target site within the plant genome (such as at or near a gene within the plant genome). Alternatively, the donor template may contain an insert sequence that does not contain a coding or transcribed DNA sequence, wherein the insert sequence is used to introduce one or more mutations into a target site within the plant genome, such as at or near a gene within the plant genome.

[0088] The donor template provided herein may contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten gene or transgenic and / or transcribed DNA sequences. Alternatively, the donor template may not contain gene, transgenic, or transcribed DNA sequences. Without limitation, the gene / transgenic or transcribed DNA sequences of the donor template may include, for example, insecticide resistance genes, herbicide tolerance genes, nitrogen use efficiency genes, water use efficiency genes, yield enancing genes, nutritional quality genes, DNA-binding genes, selection marker genes, RNAi or repression constructs, genome editing reagents, single guide RNA of a CRISPR / Cas9 system, geminivirus-based expression cassettes, or plant virus expression vector systems. According to other embodiments, the inserted sequence of the donor template may contain a protein-coding sequence or a transcribed DNA sequence encoding a non-coding RNA molecule that can target an endogenous gene for repression. The donor template may contain a promoter operatively linked to a coding sequence, gene, or transcribed DNA sequence, such as a constitutive promoter, tissue-specific or tissue-preferred promoter, developmental stage promoter, or inducible promoter. The donor template may contain a leader, enhancer, promoter, transcription start site, 5'-UTR, one or more exons, one or more introns, transcription termination site, region or sequence, 3'-UTR, and / or polyadenylation signal, each operatively linked to a coding sequence encoding non-coding RNA, a guide nucleic acid, mRNA, and / or a protein, a gene (or transgene), or a transcribed DNA sequence.

[0089] According to embodiments of the present invention, a portion (insertion sequence) of a recombinant donor template polynucleotide molecule can be inserted or integrated into a desired site or locus within the plant genome via genome editing. The insertion sequence of the donor template may contain a transgene or construct, such as a protein-coding transgene encoding a non-coding RNA molecule or a transcribed DNA sequence, said non-coding RNA molecule targeting an endogenous gene for repression. The donor template may also have one or two homologous arms flanking the insertion sequence to facilitate targeted insertion events through homologous recombination and / or homologous directed repair. Each homologous arm may have at least 70%, at least 75%, at least 80%, 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 within the plant genome. According to some embodiments, a recombinant DNA donor template molecule for site-directed or targeted integration of the insert sequence and / or recombination of one or more homologous sequences into the plant genome may be co-delivered with a DNA construct or molecule encoding a genome editing reagent, wherein the insert sequence may comprise a transgene or construct, such as a transgene or transcribed DNA sequence encoding a non-coding RNA molecule that targets an endogenous gene for repression. The recombinant DNA donor template may also contain a selection or screening marker gene and / or a transgene encoding a guide nucleic acid, wherein the marker gene and the transgene encoding the guide nucleic acid may be operatively linked to a plant-expressible promoter and / or other expression regulatory element.

[0090] As used herein, a “target site” for genome editing or site-directed integration refers to a location within a plant genome containing a polynucleotide sequence that is bound to and cleaved by a genome editing agent to introduce double-strand breaks, single-strand cuts, or other modifications (such as deamination) into the nucleic acid backbone of the polynucleotide sequence and / or its complementary DNA strand within the plant genome. Target sites may contain at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 29, or 30 consecutive nucleotides. A “target site” for directing nucleases may be a sequence contained within a double-stranded nucleic acid (DNA) molecule or either complementary strand of a chromosome at the target site. Site-specific nucleases can bind to target sites, such as via non-coding guide nucleic acids (e.g., but not limited to, CRISPR RNA (crRNA) or single guide RNA (sgRNA) as further described herein). The target sequence of the guide nucleic acid provided herein can be complementary to the target site (e.g., complementary to either strand of a double-stranded nucleic acid molecule or a chromosome at the target site). It should be understood that binding or hybridization of the target sequence of the guide nucleic acid to the target site may not require perfect identity or complementarity. For example, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight mismatches (or more) between the target site and the target sequence of the guide nucleic acid may be permissible. “Target site” also refers to the location of a polynucleotide sequence within the plant genome that can be bound to and cleaved by any other genome editing agent (such as a wide range of nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), etc.) without the guidance of the guide nucleic acid molecule to introduce double-strand breaks, single-strand cuts, or other modifications into the polynucleotide sequence and / or its complementary DNA strand.

[0091] As used herein, a “target region” or “target site” refers to a polynucleotide sequence or region flanked by two or more target sites. In some embodiments, without limitation, the target region may undergo mutations, base modifications, deletions, insertions, or inversions after double-strand breaks or nicks are repaired at the two target sites. As used herein, when describing a target region of a polynucleotide sequence or molecule, “flanked” refers to two or more target sites of a polynucleotide sequence or molecule surrounding the target region, with one target site on each side of the target region.

[0092] This document provides methods for preparing transgenic or genome-edited plants, plant parts, and seeds by delivering a DNA construct or molecule encoding a genome-editing agent into at least one cell of a mature and / or dried explant, as well as various culture and treatment steps for developing or regenerating genome-edited or transgenic plants as described herein. Transgenic or genome-edited plants, plant parts, and seeds prepared according to the methods of this invention are also provided. According to one aspect of this disclosure, development or regeneration of plants or their progeny from explants bombarded or transformed with a DNA construct or molecule encoding a genome-editing agent can be screened or selected based on markers, traits, or phenotypes resulting from editing or mutation, or from site-directed integration of inserted sequences, transgenes, etc., into developed or regenerated plants or their progeny, based on DNA constructs or molecule encoding a genome-editing agent. If a given mutation, edit, trait, or phenotype is recessive, it may be necessary to generate plants homozygous for editing or mutation through one or more generations or hybridizations (e.g., self-pollination) from an initial R0 plant so that the trait or phenotype can be observed. The conjugation of offspring plants (such as plants grown from R1 seeds or offspring) can be tested using any known conjugation assay that allows differentiation between heterozygous, homozygous, and wild-type plants (such as by using SNP assays, DNA sequencing, thermal amplification or PCR, and / or Southern blotting).

[0093] In other embodiments, screening or selection can be based on molecular assays that detect the presence of edits or mutations, or site-directed integration of inserted sequences, transgenes, etc., into plants, or their progeny plants, or one or more tissues or cells of the aforementioned plant parts or seeds that have developed or regenerated from explants bombarded or transformed with DNA encoding genome editing reagents. Assays that can be used to detect the presence of edits or mutations or transgenes introduced through site-directed integration include, for example: molecular biological assays, such as Southern and Northern blotting, PCR, FLA, and DNA sequencing; and biochemical assays, such as those by immunological means (ELISA and Western blotting) or by enzyme function or in vitro analysis to detect the presence of protein products. Alternatively, screening or selection can be based on phenotypes or traits into plants, or their progeny plants or seeds, that have developed or regenerated from explants bombarded or transformed with DNA encoding genome editing reagents, where the phenotype or trait may be a desired or predicted phenotype or trait.

[0094] IV. Definition The following definitions are provided to define and clarify the meaning of these terms with reference to relevant embodiments of this disclosure as used herein, and to guide those skilled in the art to understand this disclosure. Unless otherwise stated, the terms should be understood according to their conventional meaning and usage in the relevant fields, particularly in molecular biology and plant transformation.

[0095] An "embryo" is a part of a plant seed that consists of precursor tissues (e.g., meristems) that can develop into all or part of an adult plant. An "embryo" may also include a part of a plant embryo.

[0096] "Meristem" contains undifferentiated cells or meristematic cells that are capable of differentiating to produce all or part of one or more types of plant parts, tissues, or structures, such as buds, stems, roots, leaves, seeds, etc.

[0097] As used herein, the term "genome editing reagent" refers to any enzyme that can modify 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, the genome editing reagent includes endonucleases, recombinases, transposases, deaminases, helicases, and any combination thereof. In some embodiments, the genome editing reagent is a sequence-specific nuclease.

[0098] In one aspect, the genome editing reagent is a nuclease selected from the following: a wide range of nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), Argonaute (non-restricted examples of Argonaute proteins include *Thermophilus Argonaute* (TtAgo), *Thermococcus fibrillans* Argonaute (PfAgo), and *Haloxybacterium glabripennis* (NgAgo)), and directing nucleases such as CRISPR-associated nucleases (non-restricted examples of CRISPR-associated nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, and Cpf1). (also known as Cas12a), Csy1, Csy2, Csy3, Cse1, Cse2, 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, their homologs or their modified forms).

[0099] In some embodiments, the genome editing reagent comprises a DNA-binding domain operatively linked to a deaminase. In some embodiments, the DNA-binding domain is derived from a CRISPR-associated protein. In some embodiments, the genome editing reagent comprises uracil DNA glycosylase (UGI). In some embodiments, the deaminase is a cytidine deaminase. In some embodiments, the deaminase is an adenine deaminase. In some embodiments, the deaminase is an APOPEC deaminase. In some embodiments, the deaminase is an activation-induced 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 inactivated Cas9 nuclease, a catalytically inactivated Cpf1 nuclease, a Cas9 nickase, or a Cpf1 nickase.

[0100] In some embodiments, the genome editing reagent is a recombinase. Non-limiting examples of recombinases include tyrosine recombinases ligated to the DNA recognition motif provided herein, selected from the group consisting of Cre recombinase, Gin recombinase, Flp recombinase, and Tnp1 recombinase. In one aspect, the Cre or Gin recombinase provided herein is tethered to a zinc finger DNA-binding domain, or a TALE DNA-binding domain, or a Cas9 nuclease. In another aspect, the serine recombinase ligated 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 aspect, the DNA transposase ligated to the DNA-binding domain provided herein is selected from the group consisting of TALE-piggyBac and TALE-Mutator.

[0101] The term "regeneration" refers to the process of growing or developing a plant from one or more plant cells through one or more culture steps.

[0102] The term "recombination" in relation to polynucleotide (DNA or RNA) molecules, proteins, constructs, vectors, etc., refers to polynucleotide or protein molecules or sequences that are artificial and not normally found in nature, and / or exist in situations where such molecules or sequences are not normally found in nature. This includes polynucleotide (DNA or RNA) molecules, proteins, constructs, etc., containing combinations of two or more polynucleotide or protein sequences that would not naturally coexist without human intervention, such as polynucleotide molecules, proteins, constructs, etc., containing at least two operatively linked but heterologous polynucleotide or protein sequences. For example, the term "recombination" can refer to any combination of two or more DNA or protein sequences in the same molecule (e.g., plasmid, construct, vector, chromosome, protein, etc.), where such combinations are artificial and not normally found in nature. As used in this definition, the phrase "not normally found in nature" means not existing in nature without human introduction. Recombinant polynucleotide or protein molecules, constructs, etc., may contain (i) one or more polynucleotide or protein sequences that are isolated from each other in nature, and / or (ii) one or more polynucleotide or protein sequences that are adjacent to (or contiguous with) each other in a non-naturally adjacent manner. Such recombinant polynucleotide molecules, proteins, constructs, etc., may also refer to polynucleotide or protein molecules or sequences that have been genetically engineered and / or constructed extracellularly. For example, recombinant DNA molecules may contain any engineered or artificial plasmids, vectors, etc., and may include linear or circular DNA molecules. Such plasmids, vectors, etc., may contain various maintenance elements, including prokaryotic origins of replication and selection markers, and one or more transgenes or expression cassettes that may be present in addition to plant selection marker genes.

[0103] The term "operable link" refers to a functional link between a promoter or other regulatory element and an associated transcribed DNA sequence or coding sequence of a gene (or transgene), such that the promoter or the like operates or functions to initiate, assist, influence, induce, and / or promote the transcription and expression of the associated transcribed DNA sequence or coding sequence at least in one or more cells, tissues, developmental stages, and / or conditions.

[0104] As commonly understood in the art, the term "promoter" generally refers to a DNA sequence containing an RNA polymerase binding site, a transcription start site, and / or a TATA box that assists or promotes the transcription and expression of an associated transcribed polynucleotide sequence and / or gene (or transgene). Promoters can be artificially generated, modified, or derived from known or naturally occurring promoter sequences or other promoter sequences. Promoters may also include chimeric promoters comprising combinations of two or more heterologous sequences. Therefore, the promoters of this disclosure may include variants of promoter sequences that are compositionally similar but not identical to one or more other promoter sequences known or provided herein. Promoters can be classified according to various criteria relating to the expression pattern of the associated coding or transcribed sequence or gene (including transgene) that operatively connects the promoter, such as constitutive, developmental, tissue-specific, inducible, etc. Promoters that drive expression in all or most tissues of a plant are called "constitutive" promoters. Promoters that drive expression during certain periods or stages of development are called "developmental" promoters. Promoters that drive enhanced expression in certain tissues of a plant relative to other plant tissues are called "tissue-enhancing" or "tissue-preferred" promoters. Therefore, "tissue-preferred" promoters elicit relatively high or preferential expression in one or more specific tissues of a plant, but at lower levels in one or more other tissues of the plant. Promoters that are expressed in one or more specific tissues of a plant but are hardly expressed in other plant tissues are called "tissue-specific" promoters. "Inducible" promoters are promoters that initiate transcription in response to environmental stimuli such as cold, drought, or light, or other stimuli such as wounds or chemical application. Promoters can also be classified according to their origin, such as heterologous, homologous, chimeric, and synthetic.

[0105] As used in this article, a “plant-expressible promoter” is a promoter that can initiate, assist, influence, induce and / or promote the transcription and expression of its associated transcribed DNA sequence, coding sequence or gene in plant cells or tissues.

[0106] The term "heterologous" in relation to a related polynucleotide sequence (e.g., a transcribed DNA sequence or coding sequence or gene) refers to a promoter or other regulatory sequence that is not operablely linked to such a related polynucleotide sequence in nature without artificial introduction, for example, where the promoter or regulatory sequence has a different origin relative to the related polynucleotide sequence and / or is not naturally present in the plant species to be transformed with the promoter or regulatory sequence. Similarly, "heterologous promoter" or "heterologous plant-expressible promoter" in relation to a related polynucleotide sequence (such as a transgenic, coding sequence, or transcribed DNA sequence) means a promoter or plant-expressible promoter that is not present in the vicinity of the related polynucleotide and / or is operablely linked to the related polynucleotide in nature without artificial introduction.

[0107] In some embodiments, unless otherwise specifically indicated, the terms “a / an” and “the” as used in the context of describing a particular embodiment (especially in the context of certain claims below), and similar designations, may be interpreted to encompass both the singular and plural. In some embodiments, the term “or” is used herein to mean “and / or”, unless explicitly indicated to refer only to alternatives or that such alternatives are mutually exclusive.

[0108] The terms “comprise,” “have,” and “include” are open-ended linking verbs. Any form or tense of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes,” and “including,” is also open-ended. For example, any method that “comprises,” “has,” or “includes” one or more steps is not limited to having only those steps and may also cover other steps not listed. Similarly, any composition or apparatus that “comprises,” “has,” or “includes” one or more features is not limited to having only those features and may cover other features not listed.

[0109] The terms “percentage of identity,” “identity %,” or “percentage identical” as used herein with respect to two or more nucleotide or protein sequences are calculated as follows: (i) comparing two best-aligned sequences over a comparison window; (ii) determining the number of positions in the two sequences where the same nucleic acid base (for nucleotide sequences) or amino acid residue (for proteins) occurs to obtain the number of matching positions; (iii) dividing the number of matching positions by the total number of positions in the comparison window; and (iv) multiplying this quotient by 100% to obtain the percentage of identity. If the “percentage of identity” is calculated relative to a reference sequence without specifying a particular comparison window, the percentage of identity is determined by dividing the number of matching positions in the alignment region by the total length of the reference sequence. For example, a “comparison window” may be defined as an alignment region, in which case the “percentage of identity” is also referred to as the “alignment identity percentage.” Therefore, as used in this paper, when performing the best alignment of two sequences (query and target) (allowing gaps in their alignment), the “identity percentage” of the query sequence is equal to the number of identical positions between the two sequences divided by the total number of positions of the query sequence over its length (or comparison window), and then multiplied by 100%.

[0110] According to some embodiments, the particle complex or composition and formulation may contain an "effective amount" or "effective concentration" of a DNA construct or molecule of a coding site-specific nuclease, possibly together with other components, to edit the plant genome. The effective amount or concentration of the particle composition or formulation can depend on many factors, such as the type, size, and amount of particles used in a pre-assembled nuclease composition or formulation; the desired efficiency of genome editing; the identity and amount of other components in the composition or formulation; the specific plant species; the type of plant material used (e.g., dry-cut explants, wet-cut embryos, etc.); and the specific conditions under which the composition or formulation is applied to the plant material (e.g., temperature, culture conditions, etc.).

[0111] Some embodiments of the composition may also include an agriculturally acceptable carrier or material in combination with the particle composition. As used herein, the term “agriculturally acceptable” with respect to a carrier or material means that, as appropriate (i) it is compatible with other components of the particle / nuclease composition, at least for the purpose of using the particle / nuclease composition; (ii) it may be included in the particle composition to effectively and practically deliver the particle composition to plant material (e.g., dry explants); and (iii) it is harmless to the plant material to which the composition will be applied (at least in terms of the manner and amount in which it is applied to or associated with the plant material).

[0112] Unless otherwise stated herein or clearly contradicted by the context, all methods described herein may be performed in any suitable order. Any and all instances or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein are intended only to illustrate this disclosure and, unless otherwise requested, do not limit the scope of this disclosure.

[0113] Having described this disclosure 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 understood that all embodiments including the following in this disclosure are provided as non-limiting examples.

[0114] Example Example 1. Preparation of beads and carrier sheets for bombarding soybean explants The following is an example of a protocol for preparing beads and carrier sheets for bombarding explants. Particles or beads and carrier sheets were prepared by bombarding dry excised embryonic explants from soybean seeds using a PDS1000 helium particle gun. 50 mg of gold or tungsten particles were weighed into a clean DNase / RNase-free tube. After ultrasonic washing with 1 ml of 100% ethanol, the particles were pelleted by brief centrifugation, and the ethanol was removed. The particles were resuspended in 1 ml of 100% ethanol and stored at -20°C for later use. Before use, the particles were resuspended by sonication. 42 µl of gold or tungsten particles were transferred to a new tube and pelleted by centrifugation, and the ethanol was removed. 500 µl of sterile water was added, and the particles were resuspended by sonication. The particles were pelleted by centrifugation, and the water was removed. 25 µl of water was added to the tube, and the particles were washed with a pipette tip and then resuspended by sonication.

[0115] In soybean, there are two PDS genes, GmPDS11 (Glyma.11G253000, SEQ ID NO: 1) and GmPDS18 (Glyma.18G003900, SEQ ID NO: 2), located on chromosomes 11 (chr11) and 18 (chr18), respectively. Figure 1A As shown, three recombinant DNA constructs were designed to target the conserved regions of GmPDS11 and GmPDS18, and each recombinant DNA construct contained a transcribed DNA sequence encoding one of the guide RNAs (gRNA1, gRNA2, or gRNA3 (SEQ ID NO: 3-5)). Figure 1B As shown, recombinant DNA molecules or fragments were prepared, containing an expression cassette or transgene encoding Cas9 under a strong constitutive promoter and a transcribed DNA sequence encoding one of three gRNAs under the soybean U6 promoter. Figure 1B As further shown, these recombinant DNA molecules also contain expression cassettes or transgenes of GUS and aadA. The recombinant DNA molecules for Cas9, gRNA, GUS, and aadA expression were released as linear fragments from the DNA vector containing the recombinant DNA construct via NotI and I-CeuI restriction enzymes. The presence of left (LB) and right (RB) boundaries is shown because these DNA molecules or fragments were excised from the T-DNA vector, but since these fragments were delivered to the explant cells via particle bombardment, this should not affect these experiments.

[0116] Add the recombinant DNA molecule, construct, or fragment, possibly along with one or more other recombinant DNA molecules and / or non-coding RNA, to a tube (e.g., approximately 2.6 µg of DNA). Shortly 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. Shortly after bringing the mixture to volume, add 250 µl of ice-cold 2.5 M CaCl2 solution and 50 µl of sterile 0.1 M spermidine. Then mix this solution by low-speed vortexing. Incubate the tube on ice for at least 45 minutes to allow for particle coating. Mix the solution every 5 to 10 minutes for better results in some experiments. Pelletize the particles by low-speed centrifugation, for example, using an Eppendorf 5815 microcentrifuge at 800–1000 rpm for 2 minutes. Wash the pellets with 1 ml of ethanol and then wash the particles with a pipette tip and pelletize by centrifugation. Remove the ethanol and add 36 µl of 100% ethanol to resuspend the particles by low-speed vortexing. Use 5 µl of this preparation for each helium particle gun bombardment. For the electron gun (Accell), this preparation can be modified by combining ten 36 µl beads / particles in a scintillation vial and adding 100% EtOH to produce a final volume of 20 ml.

[0117] The above sonication step can be performed at 45-55 kHz for 1 min; the centrifugation step before coating the beads can be performed at 5000 rpm (2300 g) for 10 seconds on an IEC microcentrifuge; and the centrifugation step after DNA coating the beads can be performed at 1000 rpm (100 g) for 2 minutes on an IEC microcentrifuge.

[0118] For particle bombardment, after washing three times with sterile water, the required 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 described above were added to the particles, and the mixture was gently mixed on ice for at least 10 minutes. The coated tungsten particles were then pelleted in a microcentrifuge at 8000 xg for 30 seconds, and the supernatant was removed. The pellets were resuspended in 180 µl of sterile water by brief sonication. Shortly after sonication, the coated particles were loaded onto 6x macrocarrier membranes (30 µl each) and allowed to air dry for approximately 2 to 3 hours.

[0119] Example 2. Pre-culture of soybean explants for particle bombardment The following is an example of a protocol for preculturing explants for bombardment. Dry-cut soybean embryo explants are precultured prior to particle bombardment. Mature embryo explants are removed from dry soybean seeds as generally described, for example, in U.S. Patent No. 8,362,317. The explants are weighed for bombardment, rehydrated for 1 hour in 20% PEG4000 (Lynx 3017; see, for example, U.S. Patent Application Publication No. 2016 / 0264983) or 10% sucrose medium, and thoroughly rinsed. Lynx 1595 medium (see, for example, U.S. Patent Application Publication No. 2016 / 0264983) (or Lynx 1595 with 30 ppm Clean) can also be used in this step. Approximately 50 explants are precultured per plate on EJW 1 or EJW 2 medium (see, for example, U.S. Patent Application Publication No. 2016 / 0264983). The EJW (LIMS 4859) medium also uses TDZ levels ranging from approximately 0.5 ppm to 2 ppm. Explants are pre-cultured at 28°C in a 16 / 8 hour light cycle or in the dark for 1 to 2 days. Explants can also be pre-cultured for approximately 3 days.

[0120] Example 3. Particle bombardment of explants The following procedure can be used with the PDS1000 helium particle gun. Sterilize the gun assemblies (such as the stop screen, rupture disk, and macrocarrier holder) for approximately 1 min using 70% EtOH (or isopropanol for the carrier sheets). Load the rupture disk (e.g., a disk used in the range of approximately 650 to 2200 psi, including, for example, a 1350 psi disk) into the rupture disk retaining cap and screw it into the gas acceleration chamber. Place the stop screen on the brass nest. For each bombardment, dispense 5 µl of the helium gun preparation agent described above onto each carrier sheet. Air-dry the carrier sheet, then invert it and place it on top of the stop screen or retaining screen on the brass nest. Assemble the macrocarrier launch assembly and place it directly below the rupture disk. The gap between the rupture disk and the macrocarrier launch assembly should be approximately 1 cm.

[0121] Pre-cultured soybean explants were placed on target plate medium #42 (TPM42) and burst, with meristems facing center upwards. TPM42 medium was prepared by measuring 2 liters of distilled water into a 4 L beaker, adding 16 g of washed agar, and then autoclaving for 25 minutes to allow the agar to infiltrate the solution. TPM42 can contain 8% carboxymethyl cellulose (CMC) (for low viscosity) (or 2% CMC (for high viscosity)) and 0.4% washed agar. The solution was slightly cooled and poured into a 4 L mixer, then 320 g of CMC (low viscosity) or 80 g of CMC (high viscosity) and 2 L of water were added. The mixture was blended and transferred to 4 L plastic beakers, then autoclaved for 30 minutes, mixed, and aliquoted into four 1 L bottles. The TPM42 solution was then autoclaved again for 25 minutes and cooled to approximately 60°C before being poured into plates. Each 60 mm plate can be filled with approximately 12 to 15 ml to make approximately 300 target plates, which can be stored at 4°C or -20°C.

[0122] The following is an example of a scheme using the ACCELL electron particle gun. The bead preparation agent is heated to room temperature and vortexed. 0.5 Mil 3.2 cm 2 Polyester film (mylar) sheets are placed on small plastic dishes, optionally in a dehumidifier unit, and 320 µl of bead-forming agent is placed on the sheets. Each sheet is air-dried. Pre-cultured soybean explants are placed on TPM42 plates with the meristem facing center-up. Due to inconsistent energy of the first burst, a blank burst is performed first. A target is placed above a fixation screen, which is positioned directly above the carrier sheet. A 10 µL water droplet is vaporized by discharging a capacitor at 17.5 to 20 kV under partial helium vacuum (13.5 inHg). The shock wave generated by the vaporized droplet propels the sheet into the fixation screen, thus blocking most of the polyester film but allowing the gold beads to enter the soybean explant meristem. Between bursts, a drop of mineral oil is suspended between each point and then removed for cleaning. As before, 10 µL of water is suspended between each point. The arc chamber is covered with a PVC block, the polyester film sheet is placed over the square opening, and a screen hood is placed above the sheet and the point. Align the screen above the slide. Invert the target dish over the fixed screen so that the meristem is oriented above it, and place the weight on the dish. Cover the apparatus with a bell jar and evacuate. After 15 seconds, the vacuum reading is 13.5 in Hg, and the gun is removed.

[0123] Example 4. Culture of explants after particle bombardment The bombarded explant surfaces were plated onto EJW 1 medium and incubated overnight (other pre-culture media may also be used). In one embodiment, the plates were incubated at 28°C with a 16 / 8 photocycle. The explant surfaces were plated or embedded on B5 medium (LIMS 3485 with modified spectinomycin levels; see, for example, U.S. Patent Application Publication No. 2016 / 0264983) containing 50 to 500 ppm spectinomycin and kept at 28°C and a 16 / 8 photocycle throughout the regeneration process. In one embodiment, 250 ppm spectinomycin in B5 medium was used. The presence of the aadA selectable marker gene provides resistance or tolerance to spectinomycin as a selectant. A 24.5 g B5 custom medium mixture consisted of 3.21 g Gamborg's B5 medium, 20 g sucrose, and 1.29 g calcium gluconate. The cultures were monitored for shoot / greening and subcultured as needed.

[0124] Example 5. Identification of PDS mutants edited after particle delivery and regeneration Each emission will be 0.02 or 0.04 pmol. Figure 1B The equivalent of the DNA fragment shown was loaded onto the particle for use in biological projectile delivery. The presence of the aadA marker was screened in plants regenerated from bombarded explants. Further testing was conducted to determine whether editing was present in plants positive for the aadA marker gene, as determined by real-time quantitative PCR, due to co-delivery of recombinant DNA expressing Cas9 with gRNA targeting the PDS gene locus. Two weeks after bombardment, genomic DNA was extracted from leaf samples of the regenerated plantlets, and fragment length analysis (FLA) was used to detect the presence of editing at one or two PDS loci. FLA is a PCR-based molecular analysis that compares changes in PCR fragment length to the amplicon of a wild-type reference to identify samples with one or more mutations relative to the wild-type reference. 5' FAM-tagged primers, standard primers, and Phusion were used according to the manufacturer's instructions. TM PCR reactions were performed using polymerase (Thermo Fisher Scientific) to generate PCR fragments ranging from 200 to 500 bp. FLA primers for the GmPDS gene, as shown in SEQ ID Nos. 6 and 7, produced a 428 bp PCR fragment targeting GmPDS11 and a 384 bp PCR fragment targeting GmPDS18. PCR fragments of different sizes were considered mutations or allele edits. As shown in Figure 1, editing frequencies ranging from 17.6% to 42.5% were observed at the GmPDS locus after bombardment.

[0125] Table 1. Mutation frequency at the GmPDS locus under different Cas9 / gRNA combinations.

[0126] Example 6. Identification of site-directed integration (SDI) events at the PDS locus on chromosome 18.

[0127] Delivering Cas9 and gRNA using a donor template containing the insert sequence can guide site-directed integration (SDI) of the insert sequence at the target site of the gRNA. In some cases, one or more DNA molecules or fragments encoding genome editing components (e.g., site-specific nucleases, gRNAs, and / or marker genes) can also serve as templates for site-directed integration. Even if the DNA molecule or fragment does not contain homologous sequences for homology-mediated repair, insertion of the DNA molecule or fragment can occur via non-homologous end joining (NHEJ). Example 5 above shows that by using... Figure 1B The DNA fragments provided were bombarded with particles, resulting in numerous genome edits. The genome editing components were used in this embodiment to deliver the genome editing components. Figure 1B The DNA fragments can also be inserted at the gRNA / Cas9 target cleavage site via NHEJ. In this experiment, Figure 1B Two integration events were detected at the gRNA target site in the DNA fragment.

[0128] Four PCR assays were established to detect the presence of the inserted sequence, which could be as follows: Figure 1C The experiment was conducted in two different orientations. Beads were loaded with 0.02 or 0.04 pmol of the DNA fragment encoding Cas9 and gRNA2 from Example 5 (SEQ ID NO: 4) for each emission. 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 using primers 4005 (SEQ ID NO: 10) and 3966 (SEQ ID NO: 11), while the other orientation was detected using PCR with primers 3966 and 2438 and / or using primers 4005 and 3965. Figure 1C As shown in the image.

[0129] Events #48 and #20, which identified insertions containing at least a portion of a DNA fragment, were detected by PCR (see [link to relevant documentation]). Figure 2This was further confirmed by sequencing of the integration. Event #20 had a complete NotI sequence at one end of the insertion, but the other I-CeuI end of the fragment was not detected by PCR, indicating that a deletion or structural change at this end prevented PCR amplification. Event #20 occurred at the PDS locus on chromosome 18, and Taqman analysis further revealed that more than four complete or partial copies of the DNA fragment were integrated at this site. Event #48 was identified as having a deletion at one end of the insertion (the I-CeuI end), as detected by PCR using primers 3966 and 4005. The other end of the insertion in Event #48 was not detected by PCR, indicating that a deletion or structural change at this end prevented PCR amplification. Similar to Event #20, Event #48 occurred at the PDS locus on chromosome 18, and Taqman analysis further revealed that more than four complete or partial copies of the DNA fragment were integrated at this site.

[0130] Table 2 provides the number and frequency of SDI events at the GmPDS (Chr18) locus. Mutations in the PDS gene can cause a white leaf phenotype, which may indicate that PDS gene editing has occurred and / or is present in those tissues.

[0131] Table 2. Identification of SDI events and observed SDI frequency.

[0132] Example 7. Delivery of Cpf1, gRNA, and ssDNA into mature seed explants LbCpf1 exhibits a preference for TTTV PAM sequences; therefore, the target site GmTS1 is selected based on the presence of an appropriate PAM sequence upstream of each target sequence. A guide RNA is designed to guide the LbCpf1 protein to the target site. Recombinant DNA for expressing the LbCpf1 protein, guide RNA, and aadA is cloned.

[0133] In addition, a 70 bp ssDNA (single-stranded DNA) template modified with 5' TEG was designed. The TEG-modified ssDNA template was ordered from Integrated DNA Technologies (IDT, Product 1184, Mod Code: / 5Sp9 / ). This template has a 10 bp signature sequence containing BamHI recognition sequences flanked by 30 bp 5' and 30 bp 3' homologous arms, respectively, designed to be identical to the DNA sequence flanked at the GnTS1 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 expressing LbCpf1 protein, crRNAs, and aadA. Specifically, TransIT-2020 was used as the coating reagent to coat 80 pmol ssDNA template and 0.8 pmol recombinant DNA onto 0.6 μm gold particles (Bio-Rad; approximately 66 μg / emission). The mixture was kept on ice for >=15 min, with gentle mixing every 5 min. The coated gold particles were pelleted by brief centrifugation, and the supernatant was removed. The coated gold particles were resuspended and washed with 1 mL of pre-chilled 100% ethanol, followed by brief centrifugation to remove the ethanol. The coated gold particles were resuspended in 30 μL of pre-frozen 100% ethanol, then loaded onto microcarrier disks (5 μL per emission) and dried for 10 min.

[0134] Soybean embryo explants that had been dried and excised were rehydrated for 1 h in LIMS 3990 (B5 custom medium containing 1 g / L KNO3, 0.03 g / L Clearys 3336 WP, 3.9 g / L MES, 30 g / L, pH 5.6), thoroughly washed with sterile H2O, and cultured for 1 day in LIMS 4859 medium at 28°C and a 16 / 8 hour photoperiod. These pre-cultured mature soybean embryo explants were then bombarded according to Example 3. After bombardment, the embryo explants were transferred to LIMS 4859 medium and cultured in the dark at 28°C or 37°C for two days.

[0135] As shown in Table 3, at 28°C, the mutation rate (small insertions or deletions near the designed Cpf1 cleavage site) was 41.7% across 15 experiments, and the perfect template editing rate (with a 10 bp signature sequence containing a BamHI-recognized sequence flanking the 5' and 3' junctions defined by the homologous arms) was 0.25%. At 37°C, the mutation rate (small insertions or deletions) was 49.9% across 15 experiments, and the perfect template editing rate (with a 10 bp signature sequence containing a BamHI-recognized sequence flanking the 5' and 3' junctions defined by the homologous arms) was 0.35%.

[0136] Table 13. The effect of temperature on editing rate

[0137] While the 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 as set forth in the appended claims. Furthermore, it should be understood that although embodiments of the invention have been shown, all embodiments in this disclosure are provided as non-limiting examples and therefore should not be construed as limiting the aspects thus shown. All references cited in this disclosure are incorporated herein by reference in their entirety. The invention is intended to have the full scope defined by the language of this disclosure, the following claims, and any equivalents thereof. Therefore, the drawings and detailed description should be considered illustrative and non-limiting.

Claims

1. A method for editing a plant genome, comprising: a) Delivery of a recombinant DNA construct containing a sequence encoding a site-specific nuclease to a mature plant embryo explant, wherein the sequence is operatively linked to a plant-expressible promoter; and b) Regenerating a plant from the mature plant embryo explant, wherein in the genome of at least one cell of the regenerated plant, the regenerated plant contains editing or site-specific integration at or near the target site of the site-specific nuclease.

2. The method of claim 1, wherein the recombinant DNA construct is delivered to the mature plant embryo explant via bacterial-mediated transformation.

3. The method of claim 2, wherein the recombinant DNA construct is delivered to the mature plant embryo explant via Agrobacterium-mediated transformation.

4. The method of claim 2 or 3, wherein the T-DNA transformation vector of the recombinant DNA construct is delivered to the mature plant embryo explant.

5. The method of claim 1, wherein the recombinant DNA construct is delivered to the mature plant embryo explant via particle bombardment.

6. The method of claim 5, wherein the particles coated or applied with the recombinant DNA construct are delivered to the mature plant embryo explant via particle bombardment.

7. The method of claim 6, wherein the particle is a tungsten, palladium, or gold particle.

8. The method of claim 6 or 7, wherein the size of the particles is between about 0.5 µm and about 1.5 µm.

9. The method of claim 8, wherein the particle size is about 0.6 µm, about 0.7 µm, or about 1.3 µm.

10. The method of claim 6, wherein a plurality of particles coated or applied with the recombinant DNA construct are delivered to the mature plant embryo explant via particle bombardment.

11. The method of claim 10, wherein the amount of particles delivered to the explant is between about 50 µg and about 5000 µg, or between about 50 µg and about 5000 µg, or between about 50 µg and about 2000 µg, or between about 50 µg and about 1000 µg, or between about 50 µg and about 500 µg, or between about 100 µg and about 500 µg.

12. The method of any one of claims 1 to 11, further comprising: c) Identify regenerated plants having at least one cell containing the edited or site-specifically integrated cell at or near the target site of the site-specific nuclease.

13. The method of claim 12, wherein the identification step includes identifying regenerated plants having the edit or site-specific integration based on phenotype or trait.

14. The method of claim 12, wherein the identification step includes identifying regenerated plants having the edited or site-specific integration based on molecular assays.

15. The method of any one of claims 1 to 14, wherein the site-specific nuclease is a ribonucleoprotein.

16. The method of any one of claims 6 to 11, wherein the particle is further coated or coated with guide RNA.

17. The method according to any one of claims 1 to 16, wherein the site-specific nuclease is Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Csn1, Csx12, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Cs m4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, CasZ, or Argonaute proteins, or their homologs or modified forms.

18. The method of any one of claims 1 to 16, wherein the site-specific nuclease is the Cas9 protein.

19. The method of claim 18, wherein the Cas9 protein is derived from... Streptococcus pyogenes .

20. The method of any one of claims 1 to 16, wherein the site-specific nuclease is the Cpf1 protein.

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

22. The method of claim 21, wherein the site-specific nuclease is a broad-spectrum nuclease, a zinc finger nuclease (ZFN), a recombinase, a transposase, or a transcription activator-like effector nuclease (TALEN).

23. The method of any one of claims 1 to 22, wherein the delivery step further comprises delivering a second recombinant DNA construct or molecule to the mature plant embryo explant.

24. The method of any one of claims 6 to 11, wherein the particles are further coated or applied with a second recombinant DNA construct or molecule.

25. The method of claim 23 or 24, wherein the second recombinant DNA construct or molecule is a donor template.

26. The method of claim 25, wherein the donor template comprises a homologous sequence containing a mutation for introducing the mutation into the genome of the plant at or near the target site of a site-specific nuclease via template-mediated repair.

27. The method of claim 25, wherein the donor template comprises an insert sequence and at least one homologous sequence for integrating the insert sequence into the genome of the plant at or near the target site of the site-specific nuclease.

28. The method of claim 27, wherein the inserted sequence comprises a transgene, the transgene comprising a coding sequence or a transcribed DNA sequence operatively linked to a plant-expressible promoter.

29. The method of claim 28, wherein the transgene comprises the gene of interest.

30. The method of claim 28, wherein the transgene comprises a protein-coding sequence.

31. The method of claim 28, wherein the transgene comprises a transcribed DNA sequence encoding a non-coding RNA molecule.

32. The method of claim 28, wherein the transgene comprises a marker gene.

33. The method of claim 23 or 24, wherein the second recombinant DNA molecule comprises a marker gene.

34. The method of claim 33, wherein the marker gene is a selection marker gene.

35. The method of claim 34, wherein the selection marker gene comprises adenylate transferase (…). aadA ) gene, neomycin phosphotransferase ( nptII ) gene, hygromycin phosphotransferase ( hpt , hph or aph IV ), 5-enolpyruvate shikimate-3-phosphate synthase ( EPSPS ) gene, dicamba monooxygenase (DMO) gene or diammonium phosphate resistance ( bar ) or glufosinate N-acetyltransferase ( pat )Gene.

36. The method of claim 34, wherein the selection marker gene comprises adenylate transferase (…). aadA )Gene.

37. The method of claim 33, wherein the marker gene is a screening marker gene.

38. The method of claim 37, wherein the selection marker gene comprises a green fluorescent protein (GFP) or a β-glucuronidase (GUS) gene.

39. The method of claim 23 or 24, wherein the second recombinant DNA construct or molecule comprises a donor template region and a transgene comprising a coding sequence or a transcribed DNA sequence, wherein the transgene is located outside the donor template region of the second recombinant DNA construct or molecule.

40. The method of claim 23 or 24, wherein the second recombinant DNA construct or molecule comprises a transcribed DNA sequence encoding a guide RNA, wherein the transcribed DNA sequence is operatively linked to a plant-expressible promoter.

41. The method of any one of claims 1 to 40, wherein the recombinant DNA construct further comprises a marker gene.

42. The method of claim 41, wherein the marker gene is a selection marker gene.

43. The method of claim 42, wherein the selection marker gene comprises adenylate transferase (… aadA ) gene, neomycin phosphotransferase ( nptII ) gene, hygromycin phosphotransferase ( hpt , hph or aph IV ), 5-enolpyruvate shikimate-3-phosphate synthase ( EPSPS ) gene, dicamba monooxygenase (DMO) gene or diammonium phosphate resistance ( bar ) or glufosinate N-acetyltransferase ( pat )Gene.

44. The method of claim 42, wherein the selection marker gene comprises adenylate transferase (… aadA )Gene.

45. The method of claim 41, wherein the marker gene is a screening marker gene.

46. ​​The method of claim 45, wherein the selection marker gene comprises a green fluorescent protein (GFP) or a β-glucuronidase (GUS) gene.

47. The method of any one of claims 1 to 46, wherein the recombinant DNA construct further comprises a transcribed DNA sequence encoding a guide RNA, wherein the transcribed DNA sequence is operatively linked to a second plant-expressible promoter.

48. The method of any one of claims 1 to 46, wherein the recombinant DNA construct further comprises a donor template region.

49. The method of claim 48, wherein the donor template region comprises a homologous sequence containing a mutation for introducing the mutation into the genome of the plant at or near the target site of a site-specific nuclease via template-mediated repair.

50. The method of claim 48, wherein the donor template region comprises an insert sequence and at least one homologous sequence for integrating the insert sequence into the genome of the plant at or near the target site of the site-specific nuclease.

51. The method of claim 50, wherein the inserted sequence comprises a transgene, the transgene comprising a coding sequence or a transcribed DNA sequence operatively linked to a plant-expressible promoter.

52. The method of claim 51, wherein the transgene comprises the gene of interest.

53. The method of claim 51, wherein the transgene comprises a protein-coding sequence.

54. The method of claim 51, wherein the transgene comprises a transcribed DNA sequence encoding a non-coding RNA molecule.

55. The method of claim 51, wherein the transgene comprises a marker gene.

56. The method of any one of claims 1 to 55, further comprising: d) Select recombinant plants with a marker gene, wherein the marker gene is co-delivered with the recombinant DNA molecule.

57. The method of claim 56, wherein the marker gene is a selection marker gene.

58. The method of claim 57, wherein the selection step comprises treating the mature embryonic explant or the bud and / or root culture or plant regenerated therefrom with a selection agent.

59. The method of claim 57, wherein the selection marker gene is adenylate transferase (…). aadA )Gene.

60. The method of any one of claims 1 to 59, wherein the plant is a dicotyledonous plant.

61. The method of claim 60, wherein the plant is a soybean plant.

62. The method of any one of claims 1 to 61, wherein the mature embryonic explant comprises, prior to the delivery step, one or more of: (i) guide RNA (gRNA), (ii) a polynucleotide containing a transgene or marker gene, (iii) a polynucleotide containing a transgene encoding a non-coding RNA molecule or guide RNA, and / or (iv) a donor template.

63. The method of any one of claims 1 to 62, wherein the mature embryonic explant is a dry excision explant.

64. The method of any one of claims 1 to 63, wherein the mature embryo explant is a wet embryo explant, a dry wet embryo explant, or a wet excised embryo explant.

65. The method of any one of claims 1 to 64, wherein the moisture content of the mature embryo explant is in the range of about 3% to about 25%.

66. The method of any one of claims 1 to 65, wherein the mature embryo explant is removed from a plant seed with a moisture content ranging from about 3% to about 25%.

67. The method of claim 23 or 24, 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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