Methods of producing modified rubus

Abstract

Described herein are methods for producing a modified Rubus species plant or plant part. The method can include introducing a polynucleotide of interest (e.g., a portion of an editing system) and a gene that confers resistance to a selection agent into one or more meristem cells of a wounded tissue including meristem cells to provide a transformed tissue, culturing the transformed plant tissue in the presence of the selection agent in an amount at least 1.5 times that needed to kill 90% of Rubus spp. cells to provide a cultured transformed tissue, and regenerating a Rubus spp. plant or plant part from the cultured transformed tissue.

Description

METHODS OF PRODUCING MODIFIED RUBUSFIELD

[0001] The present invention relates to methods of producing modified Rubus plant tissue using a meristem-based transformation method that includes selection of transgenic and / or edited plant tissue.BACKGROUND

[0002] In Rubus, there are limited examples of successful Rubus transformation. In these systems, genotype, species and cultivar play a role in the transformability of plant tissues. The majority of published protocols are highly dependent on the genetic background of the cultivar, genotype, or species. In addition to the transformation step, the step of in vitro propagation is also heavily influenced by genotype or species-specific requirements. Rubus transformation is a key part in the development of products that contain consumer-valued traits, and the current state of the art is insufficient for developing a suitable and efficient system for new commercially viable genotypes.SUMMARY OF THE INVENTION

[0003] In one aspect is provided a method of producing a modified Rubus species (Rubus spp.) plant or plant part, the method comprising: wounding a Rubus spp. tissue comprising meristem cells to provide a wounded tissue comprising the meristem cells; introducing a polynucleotide of interest and a gene that confers resistance to a selection agent into one or more meristem cells of the wounded tissue to provide a transformed tissue; and culturing the transformed tissue in the presence of the selection agent in an amount at least 1.5 times that needed to kill 90% of Rubus spp. cells to provide a cultured transformed tissue; and regenerating a modified Rubus spp. plant or plant part from the cultured transformed tissue.

[0004] In another aspect, a method of producing a modified Rubus species (Rubus spp.) plant or plant part is provided that comprises: wounding a Rubus spp. tissue comprising meristem cells to provide a wounded tissue comprising the meristem cells; introducing at least a portion of an editing system and a gene that confers resistance to a selection agent into one or more meristem cells of the wounded tissue to provide a transformed tissue, wherein the portion of the editing system is targeted to a region of a gene of interest that is endogenous to the Rubus spp.; culturing the transformed tissue in the presence of the selection agent to provide a cultured transformed tissue, wherein the selection agent is in an amount at least 1.5 times thatneeded to kill 90% of Rubus spp. cells; and regenerating a modified Rubus spp. plant or plant part from the cultured transformed tissue.

[0005] In a further aspect is provided a method of producing a modified Rubus species (Rubus spp.) plant or plant part, the method comprising: wounding a Rubus spp. tissue comprising meristem cells to provide a wounded tissue comprising the meristem cells; introducing an editing system into one or more meristem cells of the wounded tissue to provide a transformed tissue, wherein the editing system is targeted (a) to a first region of a selection marker gene that is endogenous to the Rubus spp., and (b) to a second region of a gene of interest that is endogenous to the Rubus spp.; culturing the transformed tissue in the presence of a selection agent encoded by the selection marker gene to provide a cultured transformed tissue, wherein the selection agent is in an amount at least 1.5 times that needed to kill 90% of Rubus spp. cells; and regenerating a modified Rubus spp. plant or plant part from the cultured transformed tissue.

[0006] These and other aspects of the present invention are set forth in more detail in the description of the invention below.DETAILED DESCRIPTION

[0007] The present invention will now be described hereinafter with reference to the accompanying drawings and examples, in which embodiments of the invention are shown and / or discussed. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Thus, the invention contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant invention. Hence, the following descriptions are intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.

[0008] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent withtheir meaning in the context of the present application and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety for the teachings relevant to the sentence and / or paragraph in which the reference is presented. In case of a conflict in terminology, the present specification is controlling.

[0009] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a composition comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0010] As used in the description of the invention and the appended claims, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0011] Also as used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0012] The term “about” as used herein when referring to a measurable value such as an amount or concentration and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified value as well as the specified value. For example, “about X” where X is the measurable value, is meant to include X as well as variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of X. A range provided herein for a measurable value may include any other range and / or individual value therein.

[0013] As used herein, phrases such as “between X and Y” and “between about X and Y” should be interpreted to include X and Y. As used herein, phrases such as “between about X and Y” mean “between about X and about Y” and phrases such as “from about X to Y” mean “from about X to about Y.”

[0014] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as ifit were individually recited herein. For example, if the range 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed.

[0015] The term “comprise,” “comprises” and “comprising” as used herein, specify the presence of the stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0016] As used herein, the transitional phrase "consisting essentially of' (and grammatical variants) is to be interpreted as encompassing the recited materials or steps "and those that do not materially affect the basic and novel character! stic(s)" of the claimed invention. See, In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in the original); see also MPEP § 2111.03. Thus, the term "consisting essentially of as used herein should not be interpreted as equivalent to "comprising."

[0017] As used herein, the terms “increase,” “increasing,” “enhance,” “enhancing,” “improve” and “improving” (and grammatical variations thereof) describe an elevation of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500% or more such as compared to another measurable property or quantity (e.g., a control value).

[0018] As used herein, the terms “reduce,” “reduced,” “reducing,” “reduction,” “diminish,” and “decrease” (and grammatical variations thereof), describe, for example, a decrease of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% such as compared to another measurable property or quantity (e.g., a control value). In some embodiments, the reduction can result in no or essentially no (z.e., an insignificant amount, e.g., less than about 10% or even 5%) detectable activity or amount.

[0019] The term “plant part,” as used herein, includes, but is not limited to, reproductive tissues (e.g, petals, sepals, stamens, pistils, receptacles, anthers, pollen, flowers, fruits, flower bud, ovules, seeds, and embryos); vegetative tissues (e.g, petioles, stems, roots, root hairs, root tips, pith, coleoptiles, stalks, shoots, branches, bark, apical meristem, axillary bud, cotyledon, hypocotyls, and leaves); vascular tissues (e.g., phloem and xylem); specialized cells such as epidermal cells, parenchyma cells, collenchyma cells, sclerenchyma cells, stomates, guard cells, cuticle, mesophyll cells; callus tissue; and cuttings. The term “plant part” also includes plant cells, including plant cells that are intact in a plant, plant part, plant protoplast, plant tissue, plant organ, plant cell tissue culture, plant callus, plant clump, and / or the like.

[0020] As used herein, “shoot” refers to above ground (or above the media in the case ofin-vitro) (i.e., aerial) growth of a plant including a sprout and / or seedling, and optionally one or more appendages such as, a leaf, bud, stem, flowering stem, branch, and / or flower bud. In some embodiments, a “shoot” is an aerial growth that has grown above ground for about 1, 2, 3, 4, 5, 6, 7, 8, or 9 week(s) post-germination (i.e., the shoot is about 1, 2, 3, 4, 5, 6, 7, 8, or 9 week old plant tissue). In some embodiments, a “shoot” is an aerial growth that has grown above ground for about 1, 2, 3, 4, 5, 6, 7, 8, or 9 week(s) post-introduction into culture (i.e., the shoot is about 1, 2, 3, 4, 5, 6, 7, 8, or 9 week old plant culture tissue). In some embodiments, a “shoot” is an aerial growth that has grown above ground for about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 week(s) post-germination and / or post-introduction into culture. In some embodiments, a “shoot” is an aerial growth that has grown above ground for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 month(s) post-germination and / or postintroduction into culture. In some embodiments, a shoot comprises at least one meristem cell. In some embodiments, a shoot comprises a shoot meristem, a shoot apical meristem, and / or lateral meristem tissue. A shoot includes the mainstem of a plant and may have a branch and optionally a secondary branch. A branch isolated away from the rest of the plant is not considered a shoot as defined herein.

[0021] As used herein, the term “tissue culture” encompasses a culture of tissue (e.g., plant tissue), cells, protoplasts, and / or callus.

[0022] As used herein, “plant cell” refers to a cell of a plant and includes a plant cell with a cell wall and a plant cell without a cell wall such as a protoplast. In some embodiments, a plant cell is in the form of an isolated single cell or can be a cultured cell. In some embodiments, a plant cell is a part of a higher-organized unit such as, for example, a plant tissue or a plant organ. A “protoplast” is a plant cell without a cell wall or with only parts of the cell wall. Thus, in some embodiments of the invention, a transgenic cell comprising a nucleic acid molecule and / or nucleotide sequence of the invention is a cell of any plant or plant part including, but not limited to, a root cell, a leaf cell, a tissue culture cell, a seed cell, a flower cell, a fruit cell, a pollen cell, and the like. In some aspects of the invention, the plant part can be a plant germplasm. In some aspects, a plant cell can be non-propagating plant cell that does not regenerate into a plant.

[0023] “Plant tissue” as used herein means a group of plant cells organized into a structural and / or functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, explants, tissue culture and any group of plant cells assembled together to provide a structural and / or functional unit. The use of this term in conjunction with, or in the absence of, any specific type of plant tissueas listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.

[0024] An “explanf ’ as used herein refers to a plant tissue that is used as starting material for a tissue culture. In some embodiments, an explant comprises a shoot, optionally a wounded shoot or at least a portion of a shoot. In some embodiments, an explant of the invention is a fragment of a shoot. In some embodiments, an explant comprises meristem tissue (e.g., apical meristem tissue and / or lateral meristem tissue). In some embodiments, an explant comprises shoot meristem cells. In some embodiments, an explant comprises nodal meristem cells and / or tissue and / or apical shoot meristem cells and / or tissue. In some embodiments, an explant is a wounded plant tissue (e.g., a wounded shoot) that comprises meristem tissue. Meristematic tissue can be readily identified as it includes actively dividing cells that are typically small and nearly spherical. In addition, cells in the meristem have a dense cytoplasm and relatively few small vacuoles.

[0025] “Meristem” refers to undifferentiated plant tissue that includes meristem cells, which are cells that are capable of cell division and can become specialized cells. “Meristematic tissue” as used herein refers to plant tissue from the meristem or the meristem that includes meristem cells. Meristem cells and meristematic tissue can differentiate to produce one or more different plant structures such as stem, roots, leaves, germline tissue and / or seeds. A meristem may be primary or secondary. Primary meristems include the above ground meristem, referred to as the “shoot apical meristem,” and the below ground meristem, referred to as the “root apical meristem.” Secondary meristems include intercalary meristems and lateral meristems. In some embodiments, meristematic tissue of the present invention is devoid of below ground meristematic tissue (e.g., consists only of aerial meristematic tissue).

[0026] As used herein, “modified” in reference to a plant, plant part, shoot, root, tissue, or cell (e.g., a modified plant, plant part, shoot, root, tissue, or cell) refers to a plant, plant part, shoot, root, tissue, or cell, respectively, that has undergone a physical (e.g., genetic) change compared to the plant, plant part, shoot, root, tissue, or cell, respectively, at a prior time point (e.g., prior to contact with a composition of the present invention and / or prior to a method of the present invention). In some embodiments, “modified” in reference to a plant, plant part, shoot, root, tissue, or cell (e.g., a modified plant, plant part, shoot, root, tissue, or cell) refers to a plant, plant part, shoot, root, tissue, or cell, respectively, that has been subjected to genome editing, genetic transformation (e.g., introduction of a transgene), or a combination thereof. Accordingly, in some embodiments, a modified plant or plant part, shoot, root, tissue, or cell comprises a transgenic cell and / or an edited cell (i.e., a target nucleic acid of the cells has beenmodified). In some embodiments, a modified plant or plant part, shoot, root, tissue, or cell comprises a transgenic, non-edited cell and / or a transgenic, edited cell. In some embodiments, a modified plant or plant part, shoot, root, tissue, or cell comprises a non-transgenic, edited cell.

[0027] An “edited cell,” “edited plant,” “edited plant part,” “edited root,” “edited tissue,” “edited plantlet,” and / or the like as used herein refer to a cell, plant, plant part, root, tissue, plantlet, and / or the like, respectively, that comprises a modified nucleic acid in that a target nucleic acid has been modified using an editing system as described herein to provide the modified nucleic acid. Thus, an “edited cell,” “edited plant,” “edited plant part,” “edited root,” “edited tissue,” “edited plantlet,” and / or the like comprise a nucleic acid that has been modified and / or changed compared to its unmodified or native sequence and / or structure (z.e., a modified nucleic acid). The term “non-edited” refers to a condition in which a nucleic acid in the genome of a host cell or tissue or organism of interest has not been modified using an editing system as described herein.

[0028] As used herein, "modifying" or "modification" in reference to a target nucleic acid includes editing (e.g., mutating), covalent modification, exchanging / substituting nucleic acids / nucleotide bases, deleting, cleaving, and / or nicking of a target nucleic acid to thereby provide a modified nucleic acid and / or altering transcriptional control of a target nucleic acid to thereby provide a modified nucleic acid. In some embodiments, a modification may include an insertion and / or deletion of any size and / or a single base change (SNP) of any type. In some embodiments, a modification comprises a SNP. In some embodiments, a modification comprises exchanging and / or substituting one or more (e.g., 1, 2, 3, 4, 5, or more) nucleotides. In some embodiments, an insertion or deletion may be about 1 base to about 30,000 bases or more in length (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 400, 410, 420, 430,440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620,630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810,820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500,12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000,17.500, 18,000, 18,500, 19,000, 19,500, 20,000, 20,500, 21,000, 21,500, 22,000, 22,500,23,000, 23,500, 24,000, 24,500, 25,000, 25,500, 26,000, 26,500, 27,000, 27,500, 28,000,28.500, 29,000, 29,500, 30,000 bases in length or more, or any value or range therein). Thus, in some embodiments, an insertion or deletion may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 to about 310, 320, 330, 340, 350, 360,370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740,750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930,940, 950, 960, 970, 980, 990, 1000 bases in length, or any range or value therein; about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 bases to about 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460,470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650,660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840,850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 bases or more in length, or any value or range therein; about 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 bases to about 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, or 10,000 bases or more in length, or any value or range therein; or about 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, or 700 bases to about 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 bases or more in length, or any value or range therein. In some embodiments, an insertion or deletion may be about 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800,1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, or 10,000 bases to about 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 20,500, 21,000, 21,500, 22,000, 22,500, 23,000, 23,500, 24,000, 24,500, 25,000, 25,500, 26,000, 26,500, 27,000, 27,500, 28,000, 28,500, 29,000, 29,500, or 30,000 bases or more in length, or any value or range therein.

[0029] The term “transgene” as used herein refers to at least one nucleic acid sequence that is taken from the genome of one organism or produced synthetically, and which is then introduced into a host cell (e.g., a plant cell) or organism or tissue of interest and which is subsequently integrated into the host’s genome by means of “stable” transformation or transfection approaches. In contrast, the term “transient” transformation or transfection or introduction refers to a way of introducing molecular tools including at least one nucleic acid (DNA, RNA, single-stranded or double-stranded or a mixture thereof) and / or at least one amino acid sequence, optionally comprising suitable chemical or biological agents, to achieve a transfer into at least one compartment of interest of a cell, including, but not restricted to, the cytoplasm, an organelle, including the nucleus, a mitochondrion, a vacuole, a chloroplast, or into a membrane, resulting in transcription and / or translation and / or association and / or activity of the at least one molecule introduced without achieving a stable integration or incorporation into the genome and thus without inheritance of the respective at least one molecule introduced into the genome of a cell. A “transgenic” as used herein refers to a condition in which a transgene is present in the genome of a cell, tissue, or organism of interest. Thus, a “transgenic cell” refers to a cell that includes a transgene. The term “transgene-free” or “non-transgenic” refers to a condition in which a transgene is not present or found in the genome of a host cell or tissue or organism of interest.

[0030] A “mosaic” or “genetic mosaic” plant or plant tissue refers to a plant, plant part (e.g., a shoot, root, or the like), or plant tissue that is composed of cells that are not genetically identical, i.e., the plant or plant tissue has at least two different populations of cells that each have a different genotype. In some embodiments, a mosaic plant or plant tissue may comprise wild-type cells, transgenic cells (e.g., transgenic, edited cells and / or transgenic, non-edited cells), and / or edited cells (e.g., non-transgenic, edited cells and / or transgenic, edited cells). In some embodiments, a mosaic plant or plant tissue may comprise wild-type cells and transgenic cells (e.g., transgenic, edited cells and / or transgenic, non-edited cells). In some embodiments, a mosaic plant or plant tissue may comprise wild-type cells and edited cells (e.g., non- transgenic, edited cells and / or transgenic, edited cells). In some embodiments, a mosaic plantor plant tissue may be chimeric in that a portion of the cells of the plant or plant tissue include a transgene (i.e., are transgenic cells) and a portion of the cells of the plant or tissue are transgene-free (e.g., wild-type cells and / or non-transgenic, edited cells). For example, a chimeric shoot may comprise wild-type cells and transgenic cells. In some embodiments, a mosaic plant or plant tissue may comprise at least two different populations of edited cells, wherein the first population of edited cells comprises a first modified nucleic acid and the second population of edited cells comprises a second modified nucleic acid, wherein the first and second modified nucleic acids are different (e.g., a different modification was made), optionally wherein the at least two different populations of edited cells are non-transgenic.

[0031] In some embodiments, a plant or plant tissue is non-mosaic. “Non-mosaic” as used herein refers to a plant, plant part (e.g., a shoot, root, or the like), or plant tissue that is composed of cells that are genetically identical. In some embodiments, a plant tissue (e.g., a shoot) or plant comprises non-mosaic, edited cells, wherein the non-mosaic, edited cells each comprise a nucleic acid that has been modified and / or changed in the same manner compared to its unmodified or native sequence and / or structure (i.e., a modified nucleic acid) such that the non- mosaic, edited cells have the same genotype.

[0032] A “native” plant or plant part refers to a naturally occurring plant or plant part or a plant or plant part prior to a method of the present invention. In some contexts, a “native plant” plant or plant part is a plant or plant part that is non-transgenic or transgene-free and does not include a modification (e.g., a genome edit) in a nucleic acid (e.g., is non-edited) via a method of the present invention. A “native plant” plant or plant part is a plant or plant part that does not include a modification (e.g., a genome edit) in a nucleic acid (e.g., is non-edited) via a method of the present invention. A “native cell” as used herein refers to a naturally occurring plant cell or to a plant cell prior to a method of the present invention. A native cell does not include a modification (e.g., a genome edit) in a nucleic acid (e.g., is non-edited) via a method of the present invention. In some embodiments, a native plant cell is non-transgenic.

[0033] As used herein, a “wild-type” plant or plant part (e.g., a wild-type shoot, wild-type root, or wild-type cell) refers to a plant or plant part that comprises native cells in an amount greater than 99% (e.g., 99% or 100% of the cells in the plant or plant part are native cells). A “wild-type shoot” as used herein refers to a shoot that comprises native cells in an amount greater than 99% (e.g., 99% or 100% of the cells in the plant or plant part are native cells). In some embodiments, all cells of a wild-type shoot are native cells. Thus, in some embodiments, a wild-type shoot may comprise less than 1% transgenic and / or edited cells. In some embodiments, a plant is deemed to be a wild-type plant when it does not include anymodifications in the germline and / or does not pass any modifications on to its progeny. A “wild-type cell” as used herein refers to a cell that does not include a transgene that is introduced by a method of the present invention and has not been modified (e.g., a nucleic acid in the cell is not edited) via a method of the present invention. A wild-type cell is genetically identical to a native cell and may be present in a wild-type shoot and / or a transgenic root.

[0034] A "native" or "wild-type" nucleic acid, nucleotide sequence, polypeptide, amino acid sequence, or bacteria refers to a naturally occurring endogenous nucleic acid, nucleotide sequence, polypeptide, amino acid sequence, or bacteria. Thus, for example, a "native nucleic acid" is a nucleic acid that is naturally occurring in or endogenous to a reference organism. A "homologous" nucleic acid sequence is a nucleotide sequence naturally associated with a host cell into which it is introduced.

[0035] As used herein, “contact,” “contacting,” “contacted,” and grammatical variations thereof, refer to placing the components of a desired reaction together under conditions suitable for carrying out the desired reaction (e.g., transformation, transcriptional control, genome editing, nicking, and / or cleavage).

[0036] The term “transformation” or “transformed” as used herein refers to the introduction of a nucleic acid, polypeptide, and / or ribonucleoprotein (e.g., a heterologous nucleic acid, polypeptide, and / or ribonucleoprotein) into a cell (e.g., into at least one cell of a plant tissue). Transformation of a cell may be stable or transient. Thus, in some embodiments, a host cell, host tissue, or host organism may be stably transformed with a polynucleotide / nucleic acid molecule of the invention. In some embodiments, a host cell, host tissue, or host organism may be transiently transformed with a nucleic acid construct, a polypeptide, and / or a ribonucleoprotein as described herein. A “transformed plant tissue” as used herein refers to a plant tissue that comprises a cell that has been transformed (i.e., the cell comprises a nucleic acid, polypeptide, and / or ribonucleoprotein that has been introduced into the cell). In some embodiments, a transformed plant tissue may result in and / or provide at least one cell that is transgenic and not edited. In some embodiments, a transformed plant tissue may result in and / or provide at least one cell that is non-transgenic and edited. In some embodiments, a transformed plant tissue may result in and / or provide at least one cell that is transgenic and edited.

[0037] Transient transformation” in the context of a polynucleotide means that a polynucleotide is introduced into a cell (e.g., by a transformation and / or transfection approach) and does not integrate into the genome of the cell; thus, the cell is transiently transformed with the polynucleotide. A nucleic acid that is “transiently expressed” as used herein refers to anucleic acid that has been introduced into a cell and the nucleic acid is not integrated into the genome of the cell, thereby the cell is transiently transformed with the nucleic acid.

[0038] By “stably introducing” or “stably introduced” in the context of a polynucleotide introduced into a cell (e.g., by a transformation and / or transfection approach) is intended that the introduced polynucleotide is stably incorporated into the genome of the cell, and thus the cell is stably transformed with the polynucleotide. A nucleic acid that is “stably expressed” as used herein refers to a nucleic acid that has been introduced into a cell and the nucleic acid is integrated into the genome of the cell, thereby the cell is stably transformed with the nucleic acid.

[0039] Stable transformation” or “stably transformed” as used herein means that a nucleic acid molecule is introduced into a cell (e.g., by a transformation and / or transfection approach) and integrates into the genome of the cell. As such, the integrated nucleic acid molecule is capable of being inherited by the progeny thereof, more particularly, by the progeny of multiple successive generations. “Genome” as used herein includes the nuclear and the plastid genome, and therefore includes integration of the nucleic acid into, for example, the chloroplast or mitochondrial genome. Stable transformation as used herein can also refer to a transgene that is maintained extrachromasomally, for example, as a minichromosome or a plasmid.

[0040] Transient transformation may be detected by, for example, an enzyme-linked immunosorbent assay (ELISA) or western blot, which can detect the presence of a peptide or polypeptide encoded by one or more transgenes introduced into an organism. Stable transformation of a cell can be detected by, for example, a Southern blot hybridization assay of genomic DNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into an organism (e.g., a mammal, plant, etc.). Stable transformation of a cell can be detected by, for example, a northern blot hybridization assay of RNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into a host organism. Stable transformation of a cell can also be detected by, e.g., a polymerase chain reaction (PCR) or other amplification reactions as are well known in the art, employing specific primer sequences that hybridize with target sequence(s) of a transgene, resulting in amplification of the transgene sequence, which can be detected according to standard methods Transformation can also be detected by direct sequencing and / or hybridization protocols well known in the art.

[0041] “Introducing,” “introduce,” “introduced” (and grammatical variations and derivatives thereof) in the context of a polynucleotide of interest means presenting a polynucleotide of interest (e.g., polynucleotide, a nucleic acid construct, and / or an editingsystem e.g., a polynucleotide, polypeptide, and / or ribonucleoprotein)) to a host organism or cell of said organism (e.g., host cell; e.g., a plant cell) in such a manner that the polynucleotide gains access to the interior of a cell. Thus, for example, a polynucleotide from an Agrobacterium strain, such as A. tumefaciens or A. rhizogenes (that may encode or comprise at least a portion of an editing system), may be introduced into a cell of an organism, thereby transforming the cell with the polynucleotide. In some embodiments, a nucleic acid construct of the invention that encodes and / or comprises a CRISPR-Cas effector protein, a guide nucleic acid, reverse transcriptase, and / or a deaminase (e.g., a cytosine deaminase and / or adenine deaminase) may be introduced into a cell of an organism, thereby transforming the cell with the CRISPR-Cas effector protein, guide nucleic acid, reverse transcriptase, and / or deaminase. In some embodiments, a CRISPR-Cas effector protein and a guide nucleic acid may be introduced into a cell of a plant, optionally wherein the CRISPR-Cas effector protein and guide nucleic acid may be comprised in a complex (e.g., a ribonucleoprotein).

[0042] A polynucleotide may be introduced into a plant cell by any method known to those of skill in the art. In some embodiments, transformation methods include transformation via bacterial-mediated nucleic acid delivery (e.g., via Agrobacteria), viral-mediated nucleic acid delivery, silicon carbide and / or nucleic acid whisker-mediated nucleic acid delivery, liposome mediated nucleic acid delivery, microinjection, particle bombardment (e.g., microparticle bombardment), calcium-phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, nanoparticle-mediated transformation, sonication, infiltration, PEG-mediated nucleic acid uptake, as well as any other electrical, chemical, physical (mechanical) and / or biological mechanism that results in the introduction of a nucleic acid into the plant cell, including any combination thereof. In some embodiments of the invention, transformation of a cell comprises nuclear transformation. In some embodiments, transformation of a cell comprises plastid transformation (e.g., chloroplast transformation). In some embodiments, a recombinant nucleic acid construct of the invention can be introduced into a cell via conventional breeding techniques. In some embodiments, one or more of polynucleotide(s), polypeptide(s), expression cassette(s), and / or vector(s) may be introduced into a plant cell via Agrobacterium transformation. In some embodiments, one or more of polynucleotide(s), polypeptide(s), expression cassette(s), and / or vector(s) may be introduced into a plant cell via biolistic transformation. In some embodiments, one or more of polynucleotide(s), polypeptide(s), expression cassette(s), and / or vector(s) may be introduced into a plant cell via particle bombardment.

[0043] Procedures for transforming both eukaryotic and prokaryotic organisms are well known and routine in the art and are described throughout the literature (See, for example, Jiang et al. 2013. Nat. Biotechnol. 31 :233-239; Ran et al. Nature Protocols 8:2281-2308 (2013)). General guides to various plant transformation methods known in the art include Miki et al. (“Procedures for Introducing Foreign DNA into Plants” in Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E., Eds. (CRC Press, Inc., Boca Raton, 1993), pages 67-88) and Rakowoczy-Trojanowska (Cell. Mol. Biol. Lett. 7:849-858 (2002)).

[0044] A polynucleotide and / or polypeptide can be introduced into a host organism or its cell (e.g., a plant, plant part, and / or plant cell) in any number of ways that are well known in the art such as, but not limited to, by particle bombardment, transfection, a non-viral chemical method, and / or viral mediated transformation. The methods of the invention do not depend on a particular method for introducing one or more nucleotide sequences into the organism (e.g., a plant), only that they gain access to the interior of at least one cell of the organism. When more than one polynucleotide is to be introduced, it can be assembled as part of a single nucleic acid construct, as separate nucleic acid constructs, can be located on the same or different nucleic acid constructs, and / or as a complex (e.g., a ribonucleoprotein). A polynucleotide and / or polypeptide can be introduced into the cell of interest in a single transformation event, or in separate transformation events, or, alternatively, a polynucleotide and / or polypeptide can be incorporated into a plant as part of a breeding protocol.

[0045] “Root induction medium” refers to a medium that induces and / or promotes the development and / or growth of a root. In some embodiments, a root induction medium comprises a basal medium and is devoid of a growth regulator. In some embodiments, a root induction medium comprises half or quarter strength medium (e.g., half or quarter strength of that recommended by the manufacturer) and optionally a basal salt and / or vitamin. In some embodiments, a root induction medium comprises activated charcoal, a carbohydrate, a gelling agent, a growth regulator, a cytokinin, and / or an auxin. In some embodiments, a root induction medium comprises a high auxin to cytokinin ratio, e.g., an auxin to cytokinin ratio of >1. In some embodiments, a root induction medium comprises a growth regulator that comprises or consists of an auxin (e.g., a synthetic or endogenous auxin) and / or is devoid of a cytokinin. In some embodiments, a root induction medium comprises indole-3 -acetic acid (IAA), indole-3- butyric acid (IB A), 1 -naphthaleneacetic acid (NAA), 6-isopentenyladenine (IP A), 2,4- dichlorophenoxyacetic acid (2,4-D), 2-methoxy-3,6-dichlorobenzoic acid (dicamba), and / or picloram.

[0046] “Proliferation medium” or “shoot induction medium” refers to a medium that induces and / or promotes the development and / or growth of a shoot. In some embodiments, a proliferation medium comprises a basal medium. In some embodiments, a proliferation medium comprises a medium comprising a carbohydrate, a gelling agent, a growth regulator, a cytokinin, an auxin, an amino acid, casein hydrolysate, an ethylene inhibitor (e.g., silver nitrate and / or polyvinylpyrrolidone (PVP)), and / or a pH stabilizer (e.g., 2-(N- morpholino)ethanesulfonic acid (MES). In some embodiments, a proliferation medium comprises a high cytokinin to auxin ratio. In some embodiments, a proliferation medium comprises a growth regulator that comprises or consists of a cytokinin (e.g., a synthetic or endogenous cytokinin) and / or is devoid of an auxin. In some embodiments, a proliferation medium comprises 6-benzylaminopurine (BAP), zeatin, zeatin riboside, 6-(y,y- dimethylallylamino) purine (2iP), kinetin, adenine, adenine hemisulfate, and / or meta-topolin. In some embodiments, a proliferation medium comprises thidiazuron (TDZ) and / or forchlorfenuron (4-CPPU) optionally in a concentration that may cause a cytokinin-like effect due to their inhibition of cytokinin oxidase that optionally results in accumulation of endogenous cytokinin.

[0047] A “selection agent” refers to a substance (e.g., a chemical compound) whose exposure to and / or contact with favors or disfavors growth of plant cells and / or mortality of a plant cell and / or tissue. In some embodiments, a selection agent is a substance that, upon exposure to and / or contact with cells that are devoid of a gene that confers resistance to the selection agent, terminates, decreases, or suppresses the growth of all or a portion of the cells that are devoid of a gene that confers resistance to the selection agent, whereas growth is not decreased or suppressed for cells that comprise a gene that confers resistance to the selection agent upon exposure and / or contact with the substance.

[0048] A “polynucleotide of interest” may be any polynucleotide that can confer a desirable phenotype or otherwise modify the phenotype or genotype of a plant. In some embodiments, a “polynucleotide of interest” may include, but is not limited to, a polynucleotide that confers herbicide tolerance, insect resistance, nematode resistance, disease resistance, increased yield, increased nutrient use efficiency and / or abiotic stress resistance. In some embodiments, a “polynucleotide of interest” may include all or a portion of an editing system.

[0049] The present invention provides methods for producing a modified Rubus species (Rubus spp.) plant or plant part comprising wounding a Rubus spp. tissue comprising meristem cells to provide a wounded tissue comprising the meristem cells; introducing a polynucleotide of interest and a gene that confers resistance to a selection agent into one or more meristemcells of the wounded tissue to provide a transformed tissue; and culturing the transformed tissue in the presence of the selection agent in an amount at least 1.5 times that needed to kill 90% of Rubus spp. cells to provide a cultured transformed tissue; and regenerating a modified Rubus spp. plant or plant part from the cultured transformed tissue.

[0050] The present invention also provides a method of producing a modified Rubus spp. plant or plant part comprising wounding a Rubus spp. tissue comprising meristem cells to provide a wounded tissue comprising the meristem cells; introducing at least a portion of an editing system and a gene that confers resistance to a selection agent into one or more meristem cells of the wounded tissue to provide a transformed tissue, wherein the portion of the editing system is targeted to a region of a gene of interest that is endogenous to the Rubus spp.; culturing the transformed tissue in the presence of the selection agent to provide a cultured transformed tissue, wherein the selection agent is in an amount at least 1.5 times that needed to kill 90% of Rubus spp. cells; and regenerating a modified Rubus spp. plant or plant part from the cultured transformed tissue. In some embodiments, a mutation is introduced, via the editing system, into the gene of interest to provide an edited tissue.

[0051] In some embodiments, a modified Rubus spp. plant or plant part comprises transgenic and / or edited cells. In some embodiments, a modified Rubus spp. plant or plant part comprises non-transgenic, edited cells, optionally wherein the modified Rubus spp. plant or plant part is non-mosaic (e.g., the edited cells are genetically identical) or the modified Rubus spp. plant or plant part is mosaic and comprises at least two different populations of edited cells.

[0052] In some embodiments, a transformed plant tissue can be provided and / or prepared by wounding a plant tissue (e.g., a Rubus spp. plant, plant part (e.g., a shoot), or explant) comprising meristem cells to provide a wounded plant tissue (e.g., a cut tissue), and introducing a polynucleotide of interest and a gene that confers resistance to a selection agent into one or more meristem cells of the wounded tissue to provide a transformed tissue. In some embodiments, the polynucleotide of interest encodes all or a portion (e.g., at least one component) of an editing system. In some embodiments, the wounded plant tissue comprises meristem cells. In some embodiments, the wounded plant tissue comprises shoot meristem tissue and / or a node (e.g., apical meristem and / or axillary bud). In some embodiments, a transformed plant tissue can be prepared and / or provided by wounding a Rubus spp. plant tissue comprising shoot meristem cells and / or a node to provide a wounded plant tissue, and contacting the wounded plant tissue with at least one polynucleotide of interest and at least one gene that confers resistance to a selection agent. In some embodiments, a transformed planttissue can be prepared by wounding shoot meristem tissue and / or node tissue comprising meristem cells, and contacting the wounded tissue with at least one polynucleotide of interest and at least one gene that confers resistance to a selection agent, wherein the transformed plant tissue may comprise the meristem cells and one or more of the cells of the transformed plant tissue comprise the at least one polynucleotide of interest and / or the at least one gene that confers resistance to a selection agent. In some embodiments, a transformed plant tissue can be prepared by wounding plant tissue that is devoid of a root. In some embodiments, a wounded plant tissue is devoid of a root. In some embodiments, a transformed plant tissue prior to culturing on a proliferation medium and / or a root induction medium is devoid of a root and / or is devoid of a non-aerial plant part.

[0053] In some embodiments, a method of the present invention includes culturing, propagating, regenerating, and / or transforming a wounded tissue. In some embodiments, prior to wounding a plant or plant part, the method comprises collecting a plant part. For example, in some embodiments, a shoot grown from a node tissue may be collected and then blended in a blender to provide a wounded plant tissue. In some embodiments, prior to wounding a plant or plant part, the method comprises collecting Rubus spp. tissue (e.g, shoot tissue), wherein the Rubus spp. tissue comprises meristem tissue and / or a node. In some embodiments, the Rubus spp. tissue comprising meristem cells (e.g., shoot meristem cells) comprises a proliferating meristem tissue and / or a proliferating shoot. In some embodiments, a meristem cell into which a polynucleotide of interest, at least a portion of an editing system, and / or a gene that confers resistance to a selection agent is introduced is in a proliferating meristem tissue and / or a proliferating shoot. In some embodiments, a meristem cell into which a polynucleotide of interest, at least a portion of an editing system, and / or a gene that confers resistance to a selection agent is introduced is a newly formed meristem cell (e.g., a meristem cell that is about 1, 2, 3, 4, 5, or 6 days old and / or a meristem cell in a tissue that has developed within about 1, 2, 3, 4, 5, or 6 days). In some embodiments, a meristem cell into which a polynucleotide of interest, at least a portion of an editing system, and / or a gene that confers resistance to a selection agent is introduced is a meristem cell in a tissue formed within about 1, 2, 3, 4, 5, 6, 7, 8, or 9 week(s).

[0054] In some embodiments, a wounded plant tissue may be obtained by collecting one or more node(s) from a Rubus spp. plant (e.g., a blackberry or raspberry plant), optionally a greenhouse grown plant, optionally sterilizing the node(s), wounding (e.g., cutting) the node(s) by cutting (e.g., by hand) or by blending in a blender to provide wounded node tissue optionally having a smaller size than the size of the node(s) prior to wounding, and culturing the woundednode tissue optionally on solid media for about 1 week to about 6 weeks or more. During the culturing step, the wounded node tissue may produce a plant tissue comprising one or more shoot(s) (e.g., about 10 to about 20 shoots per wounded node).

[0055] In some embodiments, a method may comprise collecting an explant and / or wounding a plant tissue in a blender or by cutting (e.g., by hand manipulation) to provide a wounded tissue (e.g., fragmented or cut tissue). Methods of wounding a plant or plant part are known in the art. In some embodiments, the wounding comprises cutting the plant or plant part with a device such as a knife or scalpel, optionally by hand (e.g., human hand manipulation). In some embodiments, the wounding comprises blending the plant or plant part in a blender to thereby provide the wounded tissue.

[0056] A “blender” as used herein refers to a mechanical device that is configured to blend, chop, dice, and / or slice material such as food. In some embodiments, the blender is powered by an electric motor (z.e., an electric blender). Exemplary electric blenders include, but are not limited to, a kitchen blender that includes a rotating blade (e.g., a metal blade such as a stainless steel blade) powered by an electric motor and a food processor that includes a blade (e.g., a cutting blade such as a metal (e.g., stainless steel) cutting blade) that is powered by an electric motor. The electric motor may be a 350-watt motor. In some embodiments, a blender used in a method of the present invention is a Cuisinart blender having model number CPB-300P1 or a blender comparable thereto and the blender may be fitted with a chopping blade. A chopping blade may also be referred to as an S blade. Exemplary chopping blades and / or S blades include, but are not limited to, a blade included in Cuisinart chopping assembly having model number CPB-300CHA or a blade comparable thereto. The blade may have a planar configuration. In some embodiments, the blade assembly provided in a blender comprises a single elongate blade that rotates about its center such that it provides an edged “fin” for chopping and / or cutting on either side of its pivot axis. In some embodiments, a blade assembly provided in a blender comprises a blade with only a single edged “fin;” the blade rotates about a fixed axis and employs only the single “fin” for chopping and / or cutting. A blender of the present invention may be attached to a vessel operably associated with the blender such as Cuisinart cup having model number CTC-16. In some embodiments, the blender used in a method of the present invention is an electric blender (e.g., a kitchen blender and / or food processor) that includes and / or is fitted with a chopping blade and / or an S blade having metal (e.g., stainless steel) fins. One or more part(s) of a blender used in a method of the present invention may be able to be sterilized and / or configured for sterilization (e.g., alcohol sterilization and / or steam sterilization). In some embodiments, the parts of a blender thatcontact a plant and / or plant part may be sterilized and / or configured for sterilization. For example, a blender used in a method of the present invention may have a vessel (e.g., a cup), blade, and / or blade assembly that can be sterilized and / or that are configured for sterilization. In some embodiments, a step of wounding a plant or plant tissue in a blender is a step in a plant propagation, regeneration, and / or transformation method. Exemplary blenders, blades, systems, and / or methods of wounding a plant or plant tissue may be as described in WO 2023 / 133479, the contents of which are incorporated herein by reference in their entirety.

[0057] In some embodiments, a callus is removed from a plant tissue prior to wounding the plant tissue. In some embodiments, a callus is not removed from a plant tissue prior to wounding the plant tissue and the plant tissue including the callus are blended. In some embodiments, about 10, 15, 20, or 25 clumps to about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 clumps of shoots (e.g., blackberry or raspberry shoots) may be blended in a blender. In some embodiments, specific plant tissue may be selected and / or isolated such as when wounded tissue is being provided by hand processing. In some embodiments, a step of selection and / or isolation of specific plant tissue is not performed and a plant material is wounded (e.g., blended) such that it includes desired plant material and non-desired plant material to provided wounded plant tissue that includes non-desired plant material and optionally the wounded plant tissue including the non-desired plant material is cultured. For example, in a method that is targeting cut stem tissue, a method providing the cut stem tissue by hand processing would cut off any leaves (e.g., non-desired plant material), but in some embodiments of the present invention an entire stem with the leaves may be blended to provide a wounded tissue.

[0058] In embodiments comprising the use of a blender, the blender may be operated at a speed from about 1, 5, 10, 15, or 20 Hz to about 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, or 380 Hz. In some embodiments, the blender may be set to and / or operated at the lowest speed setting for the blender, optionally set to and / or operated at a chop setting. In some embodiments, blending comprises blending plant material (e.g., a plant or plant part) at a speed from about 1, 5, 10, 15, or 20 Hz to about 25, 30, 35, 40, 45, or 50 Hz. The blending may be carried out for a period of time such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds or more, optionally using a pulse setting. In some embodiments, blending the plant material comprises blending the plant or plant part at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) interval(s) with each interval including a time period during which the blender is operated to blend the plant material and optionally a time period during which the blender is not operated to thereby provide a rest from blending. In some embodiments, blending the plantmaterial comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) interval(s) with each interval including about 1 second to about 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds during which the blender is operated to blend the plant material and optionally about 1 second to about 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds during which the blender is not operated. In some embodiments, the blending step and / or the time to provide the wounded tissue is carried out in about 10 seconds or less such as about 9, 8, 7, 6, 5, 4, 3, 2, or 1 second(s).

[0059] A method herein and / or wounding step (e.g., a blending step) may process about 8, 9, 10, 11, 12, 13, 14, 15, or 16 ounces to about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 ounces of plant tissue (e.g., shoot tissue or explant) to provide wounded tissue in about 10 seconds or less such as about 9, 8, 7, 6, 5, 4, 3, 2, or 1 second(s). In some embodiments, a method herein and / or wounding step may process about 35 or 40 to about 45 or 50 clumps of plant tissue (e.g., plant shoots such about 3 or 6 week old plant shoots) to provide wounded tissue in about 10 seconds or less such as about 9, 8, 7, 6, 5, 4, 3, 2, or 1 second(s). In some embodiments, a method of herein and / or wounding step may process about 35, 40, 45, or 50 clumps of plant tissue (e.g., plant shoots such about 3 or 6 week old plant shoots) to provide wounded tissue in about 10 seconds or less such as about 9, 8, 7, 6, 5, 4, 3, 2, or 1 second(s). In some embodiments, a method herein and / or wounding step may process about 5 grams to about 250 grams of plant tissue to provide wounded tissue in about 10 seconds or less such as about 9, 8, 7, 6, 5, 4, 3, 2, or 1 second(s).

[0060] In some embodiments, about 8, 9, 10, 11, 12, 13, 14, 15, or 16 ounces to about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 ounces of plant material is blended with about 5, 6, 7, 8, 9, or 10 ounces to about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 ounces of liquid (e.g., water and / or an aqueous composition optionally including a bacteria). In some embodiments, about 50 clumps of plant material (e.g., plant shoots such about 3 or 6 week old plant shoots) is blended with about 5, 6, 7, 8, 9, or 10 ounces to about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 ounces of liquid (e.g., water and / or an aqueous composition optionally including a bacteria). In some embodiments, about 50 clumps of plant material (e.g., plant shoots such about 3 or 6 week old plant shoots) is blended with about 300 mL of liquid (e.g., water and / or an aqueous composition optionally including a bacteria). In some embodiments, about 5 grams to about 250 grams of plant material is blended with about 5, 6, 7, 8, 9, or 10 ounces to about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 ounces of liquid (e.g., water and / or an aqueous composition optionally including a bacteria).

[0061] “Blending,” “blend,” and grammatical variants thereof as used herein refer to mechanically cutting (e.g., chopping, dicing, etc.) material (e.g., plant material). Blending may cut the material into smaller pieces than the size of the material prior to blending. In some embodiments, blending may include coarsely cutting and / or finely cutting material. In some embodiments, blending is configured to coarsely cut plant material and aims to limit blending the plant material to a smoothie-like consistency. In some embodiments, blending provides cut plant material (e.g., wounded plant tissue) that has a size of about 0.2 or 0.3 cm to about 0.4 or 0.5 cm as measured at the widest part of the plant material. In some embodiments, blending comprises blending a whole plant or one or more part(s) thereof (e.g., an aerial portion). In some embodiments, blending comprises blending a shoot. In some embodiments, the blending of plant material is carried out in the presence of water and / or an aqueous composition (e.g., a liquid growth media). The plant material and water and / or aqueous composition may be added into a vessel of a blender and blended together. In some embodiments, plant material and bacteria, optionally in water and / or an aqueous composition, are blended together.

[0062] In some embodiments, a polynucleotide of interest and a gene that confers resistance to a selection agent are introduced into one or more meristem cells of a wounded plant tissue to provide a transformed plant tissue. The wounded plant tissue and / or transformed plant tissue may comprise meristem cells (e.g., shoot meristem cells) and / or a shoot. In some embodiments, a polynucleotide of interest and a gene that confers resistance to a selection agent are introduced into one or more meristem cells of a wounded plant tissue to provide a transformed plant tissue, wherein the transformed plant tissue comprises meristem tissue and the polynucleotide of interest and / or the gene that confers resistance to a selection agent. In some embodiments, a polynucleotide of interest and a gene that confers resistance to a selection agent are introduced into one or more meristem cells of a wounded plant tissue to provide a transformed plant tissue, wherein the transformed plant tissue comprises meristem cells and one or more of the meristem cells comprise the polynucleotide of interest and / or the gene that confers resistance to a selection agent. In some embodiments, a polynucleotide of interest and a gene that confers resistance to a selection agent are introduced into one or more meristem cells of a wounded plant tissue to provide a transformed plant tissue, wherein the transformed plant tissue comprises shoot meristem cells and the polynucleotide of interest and / or the gene that confers resistance to a selection agent. In some embodiments, a polynucleotide of interest and a gene that confers resistance to a selection agent are introduced one or more meristem cells of a wounded plant tissue to provide a transformed plant tissue, wherein the polynucleotide of interest comprises at least a portion (e.g, one component) of an editingsystem. In some embodiments, a polynucleotide of interest, a gene that confers resistance to a selection agent, and at least a portion (e.g., one component) of an editing system are introduced one or more meristem cells of a wounded plant tissue to provide a transformed plant tissue.

[0063] In some embodiments, a method of the present invention comprises a step of introducing, contacting, and / or transforming one or more meristem cells of a wounded plant tissue with a polynucleotide of interest and a gene that confers resistance to a selection agent. In some embodiments, a method of the present invention comprises a step of introducing, contacting, and / or transforming at least a portion of an editing system and a gene that confers resistance to a selection agent into one or more meristem cells of a wounded plant tissue to provide a transformed tissue, wherein the portion of the editing system is targeted to a region of a gene of interest that is endogenous to the Rubus spp. In some embodiments, the introducing, contacting, and / or transforming step may provide transformed tissue comprising a wild-type cell and a transgenic cell. In some embodiments, the introducing, contacting, and / or transforming step may provide transformed tissue comprising a transgenic cell, optionally wherein the transgenic cell is an edited cell. In some embodiments, the introducing, contacting, and / or transforming step may provide transformed tissue comprising at least two different edited cells. In some embodiments, the introducing, contacting, and / or transforming step may comprise introducing the polynucleotide of interest, the gene that confers resistance to the selection agent, and / or the at least a portion of the editing system into one or more meristem cells by particle bombardment (biolistics). In some embodiments, the introducing, contacting, and / or transforming step may comprise contacting the wounded plant tissue comprising meristem cells with an Agrobacterium that comprises the polynucleotide of interest and the gene that confers resistance to a selection agent and / or at least a portion of an editing system, and introducing the polynucleotide of interest and gene that confers resistance to a selection agent and / or at least the portion of an editing system into a meristem cell of the wounded plant tissue. Exemplary methods for contacting and / or transforming a plant tissue (e.g., a wounded plant tissue) with an Agrobacterium are known in the art and may be used in a method herein. In some embodiments, a wounded plant tissue is contacted with an Agrobacterium, optionally the wounded plant tissue may be contacted with a composition (e.g., a suspension and / or culture) comprising the Agrobacterium. In some embodiments, a wounded plant tissue is cultured and / or cultivated with the Agrobacterium and / or a composition comprising the Agrobacterium. For example, a plant tissue may be wounded with a scalpel or blender and contacted with and / or provided in an Agrobacterium cell suspension, optionally followed by sonication treatment (e.g., for about 1 minute to about 5 or 10 minutes). In some embodiments,a method of transforming a plant tissue comprises introducing a polynucleotide of interest e.g., a transgene encoding a disease resistance protein or therapeutic protein) and a gene conferring resistance and / or the at least a portion of the editing system to a selection agent from an Agrobacterium into one or more meristem cells of a wounded plant tissue to provide a transformed plant tissue.

[0064] A bacteria used in a method of the present invention (e.g., in an introducing, contacting, and / or transformation step) may be an Agrobacterium strain such as A. tumefaciens and / or rhizogenes. In some embodiments, the Agrobacterium is an A. tumefaciens strain. In some embodiments, the Agrobacterium is an A. rhizogenes strain. An Agrobacterium may comprise a polynucleotide of interest and a gene that confers resistance to a selection agent and the polynucleotide of interest may be transfer DNA (T-DNA) from the tumor-inducing (Ti) plasmid and / or hairy root-inducing (Ri) plasmid of the Agrobacterium strain. In some embodiments, the T-DNA from the Ti plasmid and / or the T-DNA from the Ri plasmid are integrated (optionally concurrently integrated) into the genome of a plant cell (e.g., a meristem cell) to provide a transgenic plant cell. In some embodiments, T-DNA from the Ti plasmid of an Agrobacterium strain is introduced into a plant cell (e.g., a meristem cell) and encodes or comprises a polynucleotide of interest (e.g., a polynucleotide encoding or comprising all or a portion of an editing system and / or a reporter gene) and / or a gene that confers resistance to a selection agent. In some embodiments, a plant tissue e.g., a wounded plant tissue comprising meristem cells) and an Agrobacterium strain, optionally in water and / or an aqueous composition, are blended together. In some embodiments, a wounding step and introducing step are carried out simultaneously. A bacteria (e.g., an Agrobacterium strain) used in a method of the present invention may include a nucleic acid construct encoding all or a portion of an editing system (e.g., a CRISPR-Cas editing system) that is configured to modify a polynucleotide (e.g., a target nucleic acid) in a plant cell. In some embodiments, a polynucleotide of interest, a gene that confers resistance to the selection agent and / or at least a portion of the editing system are in the same construct or in two or more separate constructs. In some embodiments, a bacteria (e.g., an Agrobacterium strain) may include a gene that confers resistance to a selection agent. In some embodiments, a bacteria (e.g., an Agrobacterium strain) includes a gene that confers resistance to a selection agent and a polynucleotide that encodes all or a portion of an editing system e.g., a CRISPR-Cas editing system) that is configured to modify (e.g., edit) a polynucleotide in a plant cell. In some embodiments, a bacteria (e.g., an Agrobacterium strain) includes a gene that confers resistance to a selection agent and a polynucleotide of interest encoding a transgene. In someembodiments, blending a plant tissue in the present of an Agrobacterium may inoculate the plant tissue with the Agrobacterium and / or introduce a polynucleotide from the Agrobacterium into a cell of the plant tissue, optionally into a meristem cell of the wounded tissue.

[0065] In some embodiments, the Agrobacterium strain is a wild-type Agrobacterium strain (e.g., a wild-type Agrobacterium rhizogenes strain or a wild-type Agrobacterium tumefaciens strain). In some embodiments, the Agrobacterium strain may be devoid of and / or have knocked out a wild-type bacterial transfer protein (e.g., a wild-type Agrobacterium virulence protein). For example, an Agrobacterium strain may have VirD2 knocked out.

[0066] In some embodiments, a method herein comprises introducing a polynucleotide of interest and / or a gene that confers resistance to a selection agent into a meristem cell of a wounded plant tissue, e.g., a wounded Rubus spp. plant tissue comprising one or more meristem cells, using an Agrobacterium strain that is contacted with (optionally blended with) the plant tissue thereby providing a transformed tissue. In some embodiments, a plant tissue is blended in water and / or an aqueous composition that is devoid of an Agrobacterium strain to provide a wounded plant tissue and the wounded plant tissue is contacted with (e.g., cultured with and / or in the presence of an Agrobacterium strain, optionally to introduce a polynucleotide of interest and / or a gene that confers resistance to a selection agent into a cell of the wounded plant tissue (e.g., into one or more meristem cells of the wounded tissue) and provides transformed tissue.

[0067] In some embodiments, at least one cell of a wounded plant tissue is stably transformed with a polynucleotide from an Agrobacterium strain, optionally wherein the polynucleotide comprises a gene that confers resistance to a selection agent and / or a polynucleotide encoding or comprising a portion or all of an editing system. In some embodiments, a polynucleotide (e.g., a polynucleotide from an Agrobacterium strain and / or that encodes or comprises a portion or all of an editing system) is stably expressed in the plant cell and transient expression of the polynucleotide in the plant cell allows and / or provides for a target region of a gene of interest that is endogenous to the Rubus spp. to be modified in the plant cell (e.g., meristem cell) and / or another plant cell and thereby provides a modified nucleic acid.

[0068] In some embodiments, a plant cell is transiently transformed with a polynucleotide from an Agrobacterium strain, optionally wherein the polynucleotide comprises a gene that confers resistance to a selection agent and / or a portion or all of an editing system. In some embodiments, a polynucleotide (e.g., a polynucleotide from an Agrobacterium strain and / or that encodes or comprises a portion or all of an editing system) is transiently expressed in the plant cell and transient expression of the polynucleotide in the plant cell allows and / or providesfor a target region of a gene of interest that is endogenous to the Rubus spp. to be modified in the plant cell (e.g., meristem cell) and / or another plant cell and thereby provides a modified nucleic acid.

[0069] A method of the present invention may comprise one or more culturing steps (e.g., 1, 2, 3, 4, 5, or more), which may be carried out using methods known in the art. In some embodiments, culturing a plant tissue (e.g., a shoot material, a wounded plant tissue, transformed plant tissue, explant, and / or modified shoot) may include exposing the plant tissue to certain conditions (e.g., light, dark, nutrients, humidity, etc.) for a period of time (e.g., about 1, 2, 3, 4, or 5 day(s) to about 21, 25, 30, 35, 40, 45 or 50, or more days). In some embodiments, culturing a plant tissue comprises providing temperature and / or light conditions sufficient to maintain and / or grow the plant tissue. In some embodiments, culturing a plant tissue is carried out before and / or after a wounding step. In some embodiments, a method herein may comprise culturing a shoot material. In some embodiments, a method herein may comprise culturing a wounded plant tissue. In some embodiments, a method herein may comprise culturing a transformed plant tissue. In some embodiments, a method herein may comprise culturing a modified shoot. In some embodiments, a method herein may comprise culturing a plant tissue in vitro, e.g., via tissue culture. A plant material may be cultured in the presence of and / or on media that may include an antibiotic and / or a growth hormone (e.g., a plant growth hormone, and / or a nutrient), optionally wherein the plant material comprises (e.g., is in contact with an Agrobacterium strain).

[0070] In some embodiments, prior to wounding a Rubus spp. tissue, Rubus spp. shoot material comprising a node is collected and the shoot material comprising the node is cultured in the presence of at least one plant growth regulator (e.g., cultured for about 1, 2, 3, 4, 5, 6 or 7 weeks, or any range or value therein) to produce a Rubus spp. tissue comprising meristem cells (e.g., shoot meristem cells). In some embodiments, Rubus spp. shoot material comprising a node is cultured in the presence of at least at least one plant growth regulator. In some embodiments, Rubus spp. shoot material comprising a node is cultured in the presence of a cytokinin, a cytokinin-like compound, an auxin, an auxin precursor, and / or an antibiotic to produce a Rubus spp. tissue comprising meristem cells. In some embodiments, Rubus spp. shoot material comprising a node is cultured in the presence of a cytokinin or cytokinin-like compound selected from thidiazuron (TDZ), 6-benzylaminopurine (BAP), kinetin, forchlorfenuron (4-CPPU), 6-(y,y-dimethylallylamino) purine (2iP), zeatin, zeatin-riboside, dihydro-zeatin, adenine, 6-isopentenyladenine (IP A), and / or TIBA (2,3,5-triiodobenzoic acid). In some embodiments, Rubus spp. shoot material comprising a node is cultured in the presenceof TDZ and hygromycin to produce a Rubus spp. tissue comprising meristem cells. In some embodiments, Rubus spp. shoot material comprising a node is cultured in the presence TDZ in an amount of about 0.1 mg / L to about 5 mg / L (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 mg / L, or any range or value therein) and in the presence of hygromycin in an amount of about 1 mg / L to about 10 mg / L (e.g., about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3,4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5,8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0 mg / L, or any range or value therein).

[0071] A method and / or culturing step herein may include culturing a transformed plant tissue (e.g., transformed plant tissue comprising meristem cells comprising a polynucleotide of interest and / or a gene that confers resistance to a selection agent) in the presence of a selection agent to provide a cultured transformed tissue. While not wishing to be bound by theory, it is believed that transformed meristem cells have a higher tolerance for elevated levels of selection agent as compared to transformed non-meristematic cells or wild-type cells. In this respect, meristem cells transformed with a polynucleotide of interest and a gene that confers resistance to a selection agent can be cultured in the presence of elevated levels of selection agent. Accordingly, a method and / or culturing step herein can comprise culturing the transformed tissue in the presence of a selection agent in an amount at least 1.5 times that needed to kill 90% of Rubus spp. cells from a control Rubus tissue (e.g., the same Rubus tissue used to provide the wounded tissue) within 48 hours in the same basal medium and environmental conditions. In some embodiments, a transformed plant tissue (e.g., transformed plant tissue comprising meristem cells comprising a polynucleotide of interest and / or a gene that confers resistance to a selection agent) is cultured in the presence of kanamycin in an amount at least 1.5 times that needed to kill 90% of Rubus spp. cells from a control Rubus tissue within 48 hours. In some embodiments, a transformed plant tissue is cultured in the presence of at least about at least about 75 mg / L kanamycin, e.g., about 75, 80, 85, or 90 mg / L to about 95, 100, 105, 110, 115, or 120 mg / L kanamycin, or any value or range therein, to provide a cultured transformed tissue. In some embodiments, a transformed plant tissue is cultured in the presence of about 100 mg / L kanamycin to provide a cultured transformed tissue. In some embodiments, a transformed plant tissue (e.g., transformed plant tissue comprising meristem cells comprising a polynucleotide ofinterest and a gene that confers resistance to a selection agent) is cultured in the presence of spectinomycin in an amount at least 1.5 times that needed to kill 90% of Rubus spp. cells from a control Rubus tissue within 48 hours. In some embodiments, a transformed plant tissue is cultured in the presence of at least about at least about 150 mg / L spectinomycin, e.g., about 150, 155, or 160 mg / L to about 165, 170, or 175 mg / L spectinomycin, or any value or range therein, to provide a cultured transformed tissue. In some embodiments, a transformed plant tissue is cultured in the presence of a selection agent in an amount at least 1.5 times that needed to kill 90% of Rubus spp. cells for a period of at least about 48 hours (e.g., 48, 60, 72, 84, 96, 108 or 120 hours) to provide a cultured transformed tissue. In some embodiments, the culturing step may provide cultured transformed tissue comprising a wild-type cell and a transgenic cell. In some embodiments, the culturing step may provide cultured transformed tissue comprising a transgenic cell, optionally wherein the transgenic cell is an edited cell. In some embodiments, the culturing step may provide cultured transformed tissue comprising at least two different edited cells.

[0072] To produce a modified Rubus spp. plant or plant part, a method herein may include a step of regenerating a modified Rubus spp. plant or plant part from a cultured transformed tissue. In some embodiments, the regenerating step comprises culturing the cultured transformed tissue on a proliferation medium (e.g., shoot induction medium) to product a shoot and subsequently culturing the shoot on root induction medium to provide the modified Rubus spp. plant or plant part. In some embodiments, culturing the cultured transformed tissue on a proliferation medium comprises contacting at least a portion of the cultured transformed tissue with the proliferation medium such as by laying, placing, staking, and / or the like the cultured transformed tissue onto and / or in the proliferation medium. In some embodiments, a shoot may originate and / or form from one or more transformed meristem cell(s) of a cultured transformed tissue. A shoot originating and / or formed from a transgenic meristem cell may comprise cells that are genetically the same as the meristem cells from which the shoot was formed, e.g., at least a portion of the cells of the shoot may be modified in the same manner. Accordingly, in some embodiments, the methods herein provide for and / or produce a modified Rubus spp. shoot, wherein cells of the modified shoot comprise transgenic cells, optionally wherein the transgenic cells are edited cells. In some embodiments, all or substantially all (e.g., greater than 90%, 95%, 96%, 97%, 98%, 99% or 100% of the total number of cells) of the cells of a modified shoot of the present invention may comprise a gene that confers resistance to a selection agent. In some embodiments, all or substantially all (e.g., greater than 90% of the total number of cells) of the cells of a modified shoot of the present invention are transgene-free and comprise a modified nucleic acid. In some embodiments, all or substantially all (e.g., greater than 90% of the total number of cells) of the cells of a modified shoot of the present invention comprise a modified nucleic acid and a gene that confers resistance to a selection agent. In some embodiments, all or substantially all (e.g., greater than 90% of the total number of cell) of the cells of a modified shoot of the present invention comprise a transgene that is not a gene that confers resistance to a selection agent, e.g., a transgene encoding a therapeutic protein. In some embodiments, all or substantially all (e.g., greater than 90% of the total number of cells) of the cells of a modified shoot comprise a gene that confers resistance to a selection agent and a second transgene. In some embodiments, culturing a cultured transformed tissue on a proliferation medium produces at least two modified shoots, wherein two or more of the at least two modified shoots may be the same or may be different (e.g., comprise different edits).

[0073] A proliferation medium (e.g., shoot induction medium) of use in a method and / or culturing step of the present invention may comprise a basal medium that includes one or more (e.g., 1, 2, 3, 4, 5, or more) growth regulator(s). In some embodiments, a proliferation medium includes a basal medium and a selection agent. In some embodiments, a proliferation medium is devoid of a selection agent. The term “plant growth regulator,” as used herein, encompasses naturally occurring or synthetic (not naturally occurring) compounds that regulate and / or aid in plant growth and / or development. Exemplary plants growth regulators, include, but are not limited to, auxins or auxin precursors and cytokinins or cytokinin-like compounds. Exemplary cytokinins include, but are not limited to, thidiazuron, BAP (6-benzylaminopurine), kinetin, 4- CPPU (forchlorfenuron), 2iP (6-(y,y-dimethylallylamino) purine), zeatin, zeatin-riboside, dihydro-zeatin, adenine, 6-isopentenyladenine (IP A), TIBA (2,3,5-triiodobenzoic acid) and / or PhytoAx™ plant growth regulating preparation commercially available from PhytoTech Labs.

[0074] Upon generation of a modified shoot, the modified shoot is cultured on a root induction medium to provide the modified Rubus spp. plant or plant part. In some embodiments, a root induction medium comprises a selection agent (e.g., an antibiotic such as kanamycin or spectinomycin) and may further comprise a basal medium, an antioxidant, a micronutrient, a cytokine, a plant growth regulator, an antibiotic, and / or a phenol. In some embodiments, a root induction medium comprises a basal medium, e.g., a medium capable of supporting the development and / or growth of plant tissue optionally containing an inorganic salt, vitamin, sugar (e.g., glucose), buffer system, and / or amino acid (e.g., an essential amino acid), and the basal medium may comprise a selection agent. In some embodiments, a root induction medium comprises a basal medium, a selection agent, and an auxin or auxinprecursor. Exemplary auxins or auxin precursors include, but are not limited to natural auxins and / or natural auxin precursors, such as those obtainable from seaweed and / or algae, synthetic auxins and / or synthetic auxin precursors, and / or auxin conjugates. Examples of auxins include, but are not limited to, indole-3 -acetic acid (IAA), 4-chloro-indole-3 -acetic acid (4-C1-IAA), phenylacetic acid (PAA), indole-3 -butyric acid (IBA), indole-3-acetyl-l-O-P-D-glucose (lAAglc), 1 -naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), 2- methoxy-3,6-dichlorobenzoic acid (dicamba), 4-amino-3,5,6-trichloropicolinic acid (tordon), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2,3,6-trichlorobenzoic acid, 4-chloro-2 methyl phenoxy acetic acid (MCPA) and / or N,N-dimethylethylthiocarbamate. Auxin conjugates may also be used including, e.g., JAA-Inositol, lAA-Inositol-arabinose, IAP1, an lAA-peptide, an IAA glycoprotein, an lAA-glucan, lAA-aspartate, lAA-glucose, IAA-1-O-glucose, lAA-myo- Inositol, IAA-4-O-glucose, IAA-6-O-glucose, lAA-Inositol-galactose, an IAA amide conjugate, and / or an lAA-amino acid conjugate. In some embodiments, a root induction medium comprises a basal medium that comprises a selection agent and indole-3 -butryic acid (IBA). In some embodiments, a root induction medium comprises a selection agent and auxin or an auxin precursor, wherein the auxin or auxin precursor is present in the root induction medium at a concentration of less than about 0.5 mg / L, e.g., less than about 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1 or 0.05 mg / L. In some embodiments, an auxin or auxin precursor is present in a root induction medium at a concentration of about 0.01, 0.05, or 0.1 mg / L to about 0.2 or 0.3 mg / L. In some embodiments, the auxin or auxin precursor is present in a root induction medium at a concentration of about 0.1 mg / L. In some embodiments, a root induction medium comprises a selection agent and auxin or an auxin precursor, wherein the auxin or auxin precursor is present in the root induction medium at a concentration about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / L. In some embodiments, a root induction medium comprises a selection agent and auxin or an auxin precursor, wherein the auxin or auxin precursor is present in the root induction medium at a concentration about 1, 2, 3, 4, or 5 mg / L to about 6, 7, 8, 9, or 10 mg / L. In some embodiments, a root induction medium comprises an auxin or auxin precursor in a root induction medium and / or a dip treatment (e.g., an IBA dip treatment) at a concentration about 500, 600, 700, 800, 900, 1000, 1100, or 1200 mg / L.

[0075] In some embodiments, the presence of a selection agent in a medium is used in a method of the present invention to select a transformed and / or transgenic cell and / or tissue. In some embodiments, culturing a cultured transformed plant tissue on a medium comprising a selection agent allows for and / or provides a shoot and / or root to grow and / or develop that comprises one or more cells that include a gene that confers resistance to the selection agent,whereas growth of a wild-type shoot and / or root from the cultured transformed plant tissue cultured on medium comprising the selection agent is stunted or a wild-type root does not develop. In some embodiments, a wounded plant tissue and / or transformed plant tissue includes a gene conferring resistance to a selection agent (e.g., a resistance gene). A selection agent may be an antibiotic, herbicide and / or or aluminum. In some embodiments, the selection agent is glyphosate, bialaphos, phosphinothricin, Basta, glufosinate, 2,4-D, imazamox, kanamycin or a related aminoglycosides, hygromycin, ampicillin, or dicamba. A resistance gene that may be used in a method of the present invention includes, but is not limited to, the neomycin phosphotransferase II (NPT II / Neo) gene, which confers resistance to kanamycin and related antibiotics (Messing & Vierra (1982) Gene 19:259-268; Bevan et al. (1983) Nature 304: 184-187); the bar gene, which confers resistance to the herbicide phosphinothricin (White et al. (1990) Nucl. Acids Res. 18:1062, Spencer et al. (1990) Theor. AppL Genet. 79:625-631); the EPSPS gene, which confers resistance to glyphosate (U.S. Pat. Nos. 4,940,935 and 5, 188,642); the aminoglycoside 3”-adenyltransferase encoded by the aadA gene, which confers resistance to spectinomycin; the acetolactate synthase (ALS) gene, which confers resistance to imazamox or chlorsuluron; the 5 -oxoprolinase gene, which confers resistance to sulfonamides; and Aluminum Resistance Transcription Factor 1 (ART1) gene and / or a ZMAT6 gene, which confer resistance to aluminum. In some embodiments, the gene that confers resistance is NPT II / Neo and the selection agent is kanamycin. In some embodiments, the gene that confers resistance is aadA and the selection agent is spectinomycin. In some embodiments, the selection agent is not hygromycin.

[0076] Culturing a cultured transformed plant tissue on a proliferation medium and a root induction medium in accordance with a method of the present invention provides a modified Rubus spp. plant or plant part. A modified plant or plant part produced by a method of the present invention may include a gene conferring resistance to a selection agent, one or more other transgenes (i.e., a transgene other than the resistance gene), and / or one or more modified nucleic acid(s) (e.g., one or more edited gene(s)). Thus, in some embodiments, the modified Rubus spp. plant or plant part may comprise (i) transgenic, non-edited cells, (ii) transgenic, edited cells, or (iii) transgene-free edited cells.

[0077] A transgene that may be included in a modified plant or plant part in accordance with a method of the present invention includes, but is not limited to, a transgene encoding an agronomic trait, insect resistance, disease resistance, herbicide resistance, sterility, grain characteristics, and / or the like. A transgene may be involved in metabolism of oil, starch, carbohydrates, nutrients, etc. Thus, transgenes or traits of interest include, but are not limitedto, environmental- or stress-related traits, disease-related traits, and traits affecting agronomic performance. Transgenes may also include genes responsible for the synthesis of proteins, peptides, fatty acids, lipids, waxes, oils, starches, sugars, carbohydrates, flavors, odors, toxins, carotenoids, hormones, polymers, flavonoids, storage proteins, phenolic acids, alkaloids, lignins, tannins, celluloses, glycoproteins, glycolipids, marker proteins, etc. In some embodiments, transgenes may include those encoding enzymes responsible for oil production; enzymes that synthesize, or proteins which are themselves, natural medicaments such as pharmaceuticals or veterinary products. Such proteins may include, but are not limited to, retinoblastoma protein, p53, angiostatin, leptin, serum albumin, hemoglobin, or collagen.

[0078] In some embodiments, the modified Rubus spp. plant or plant part comprises at least one modified (e.g., edited) nucleic acid, wherein the modified nucleic acid is provided in accordance with a method of the present invention to provide the modified Rubus spp. plant or plant part, and the modified Rubus spp. plant or plant part is devoid of a transgene introduced into a plant cell in accordance with a method of the present invention to provide the modified Rubus spp. plant or plant part. In some embodiments, a modified Rubus spp. plant or plant part comprises at least one modified (e.g., edited) nucleic acid, wherein the modified nucleic acid is provided in accordance with a method of the present invention to provide the modified Rubus spp. plant or plant part, and the modified plant or plant part is transgene-free. In some embodiments, a modified Rubus spp. plant or plant part comprises at least one modified (e.g., edited) nucleic acid, wherein the modified nucleic acid is provided in accordance with a method of the present invention to provide the modified Rubus spp. plant or plant part, and optionally the modified Rubus spp. plant or plant part is transgenic. In some embodiments, the modified Rubus spp. plant or plant part is an edited plant, wherein the edited plant is produced in about 2 to about 6 months (e.g., about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5 or 6 months, or any range or value therein) after the introducing step.

[0079] In some embodiments, the invention further provides a method of producing a modified Rubus species (Rubus spp.) plant or plant part comprising wounding a Rubus spp. tissue comprising meristem cells to provide a wounded tissue comprising the meristem cells; introducing an editing system (e.g., guide, editor into one or more meristem cells of the wounded tissue to provide a transformed tissue, wherein the editing system is targeted (a) to a first region of a selection marker gene (e.g., an inactive selection marker gene) that is endogenous to the Rubus spp., and (b) to a second region of a gene of interest that is endogenous to the Rubus spp.; culturing the transformed tissue in the presence of a selection agent for which resistance to the selection agent is encoded by the selection marker gene (e.g., the edited activeselection marker gene which confers resistance; e.g., the selection marker gene is edited resulting in resistance to the selection agent) to provide a cultured transformed tissue, wherein the selection agent is in an amount at least 1.5 times that needed to kill 90% of Rubus spp. cells; and regenerating a modified Rubus spp. plant or plant part from the cultured transformed tissue. In some embodiments, a first mutation is introduced into the selection marker gene to provide an edited selection marker gene that confers resistance to the selection agent and / or a second mutation is introduced into the gene of interest to provide an edited tissue.

[0080] In some embodiments, editing of a selection marker gene (e.g., inactive selective marker gene) that is endogenous to the Rubus spp. results in an edited selection marker gene (e.g., encoding a mutant protein or gene knockout) that confers resistance to a selection agent. In some embodiments, the selection marker gene that is endogenous to the Rubus spp. includes, but is not limited to, the Multiple Antibiotic Resistance (MARI) gene, ALS gene, 5- oxoprolinase gene, ART1 gene and ZMAT6 gene. Acetolactate synthase is a key enzyme for the biosynthesis of branched chain amino acids and is the target site of several important herbicides including sulfonylureas (e.g., chlorsulfuron) and imidazolinones (e.g., imazethapyr). ALS harboring point mutations such as Prol97Ser and / or Ser653Asn substitutions can confer sufficient tolerance to herbicidal ALS inhibitors (Jander et al. (2003) Plant Physiol. 131 : 139-146; Ray et al. (2004) J. Plant Physiol. 161(9): 1079-83) and Cas9CBE- mediated gene editing has been used to generate SNPs that confer resistance in Canola (Wu et al. (2020) Plant Biotechnol. J. 18(9): 1857-1859) and Arabidopsis (Chen at al. (2017) Sci. China Life Sci. 60(5):520-523). Similarly, an Ala441Val mutation in MARI confers resistance to the aminoglycosides kanamycin, tobramycin, gentamicin, streptomycin, amikacin and apramycin (Conte et al. (2009) Plant Physiol. 151(2):559-573). CRISPR / Cas9-mediated gene editing of the Arabidopsis 5 -oxoprolinase 1 (OXPL) gene resulted in indel mutants exhibiting a reduced sensitivity to sulfamethoxazole (Baeg et al. (2021) Plant Biotechnol. Rep. 15:753- 764). In some embodiments, the endogenous selection marker gene is: (a) Multiple Antibiotic Resistance (MARL) gene and the selection agent is kanamycin; (b) an Acetolactate Synthase (ALS) gene and the selection agent is chlorsulfuron or imazamox; (c) a 5 -oxoprolinase gene and the selection agent is a sulfonamide; and / or (d) an Aluminum Resistance Transcription Factor 1 (ART1) gene and / or a ZMAT6 gene and the selection agent is aluminum. In some embodiments, a method of the present invention is devoid of editing a selection marker gene (e.g., inactive selective marker gene that is optionally endogenous) to result in an edited selection marker gene (e.g., encoding a mutant protein or gene knockout) that confers resistance to a selection agent.

[0081] Modification of a target nucleic acid (e.g., a gene of interest and / or a selection marker gene that is endogenous to the Rubus spp.) may be accomplished using an editing system. A “gene editing system” or “editing system” as used herein refers to any site-specific (e.g., sequence-specific) nucleic acid editing system now known or later developed, which system can introduce a modification (e.g., a mutation) in a nucleic acid in target specific manner. For example, an editing system (e.g., a site- and / or sequence-specific editing system) can include, but is not limited to, a CRISPR-Cas editing system, a meganuclease editing system, a zinc finger nuclease (ZFN) editing system, a transcription activator-like effector nuclease (TALEN) editing system, a base editing system and / or a prime editing system, each of which may comprise one or more polypeptide(s) and / or one or more polynucleotide(s) that when present and / or expressed together (e.g., as a system) in a composition and / or cell can modify (e.g., mutate) a target nucleic acid in a sequence specific manner. In some embodiments, an editing system (e.g., a site- and / or sequence-specific editing system) can comprise one or more polynucleotide(s) and / or one or more polypeptide(s), including but not limited to a nucleic acid binding polypeptide (e.g., a DNA binding domain), a nuclease, another polypeptide, and / or a polynucleotide. In some embodiments, a CRISPR-Cas editing system is provided and / or is used that comprises a CRISPR-Cas effector protein.

[0082] In some embodiments, an editing system comprises one or more sequence-specific nucleic acid binding polypeptide(s) (e.g., a DNA binding domain) that can be from, for example, a polynucleotide-guided endonuclease, a CRISPR-Cas endonuclease (e.g., CRISPR- Cas effector protein), a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN) and / or an Argonaute protein. In some embodiments, an editing system comprises one or more cleavage polypeptide(s) (e.g., nucleases) including, but not limited to, an endonuclease (e.g., Fokl), a polynucleotide-guided endonuclease, a CRISPR-Cas endonuclease (e.g., CRISPR-Cas effector protein), a zinc finger nuclease, and / or a transcription activator-like effector nuclease (TALEN).

[0083] A “nucleic acid binding polypeptide” as used herein refers to a polypeptide or domain that binds and / or is capable of binding a nucleic acid (e.g., a target nucleic acid). A DNA binding domain is an exemplary nucleic acid binding polypeptide and may be a site- and / or sequence-specific nucleic acid binding domain. In some embodiments, a nucleic acid binding polypeptide may be a sequence-specific nucleic acid binding polypeptide such as, but not limited to, a sequence-specific binding domain from, for example, a polynucleotide-guided endonuclease, a CRISPR-Cas effector protein (e.g., a CRISPR-Cas endonuclease), a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN) and / or an Argonauteprotein. In some embodiments, a nucleic acid binding polypeptide comprises a cleavage domain (e.g., a nuclease domain) such as, but not limited to, an endonuclease (e.g., Fokl), a polynucleotide-guided endonuclease, a CRISPR-Cas endonuclease, a zinc finger nuclease, and / or a transcription activator-like effector nuclease (TALEN). In some embodiments, the nucleic acid binding polypeptide associates with and / or is capable of associating with (e.g., forms a complex with) with one or more nucleic acid molecule(s) e.g., forms a complex with a guide nucleic acid as described herein), which may direct and / or guide the nucleic acid binding polypeptide to a specific target nucleotide sequence (e.g., a gene locus of a genome) that is complementary to the one or more nucleic acid molecule(s) (or a portion or region thereof), thereby causing the nucleic acid binding polypeptide to bind to the nucleotide sequence at the specific target site. In some embodiments, the nucleic acid binding polypeptide is a CRISPR-Cas effector protein as described herein.

[0084] In some embodiments, an editing system comprises or is a ribonucleoprotein such as an assembled ribonucleoprotein complex e.g., a ribonucleoprotein that comprises a CRISPR-Cas effector protein, a guide nucleic acid, and optionally a reverse transcriptase or a deaminase). In some embodiments, a ribonucleoprotein of an editing system may be assembled together e.g., a pre-assembled ribonucleoprotein including a CRISPR-Cas effector protein, a guide nucleic acid, and optionally a reverse transcriptase or a deaminase) such as when contacted to a target nucleic acid or when introduced into a cell e.g., a mammalian cell or a plant cell). In some embodiments, a ribonucleoprotein of an editing system may assemble into a complex (e.g., a covalently and / or non-covalently bound complex) while a portion of the ribonucleoprotein is contacting a target nucleic acid and / or may assemble after and / or during introduction into a plant cell. In some embodiments, an editing system may be assembled (e.g., into a covalently and / or non-covalently bound complex) when introduced into a plant cell. In some embodiments, a ribonucleoprotein may comprise a CRISPR-Cas effector protein, a guide nucleic acid, and optionally a reverse transcriptase. In some embodiments, a ribonucleoprotein may comprise a CRISPR-Cas effector protein, a guide nucleic acid, and optionally a deaminase.

[0085] In some embodiments, an editing system comprises a reverse transcriptase, an extended guide nucleic acid, and a CRISPR-Cas effector protein, e.g., a Type II CRISPR-Cas effector protein or Type V CRISPR-Cas effector protein. In some embodiments, the Type V CRISPR-Cas effector protein or Type II CRISPR-Cas effector protein, the reverse transcriptase, and the extended guide nucleic acid may form a complex or may be comprised in a complex that is capable of interacting with a target nucleic acid.

[0086] In some embodiments, an editing system is used in prime editing. “Prime editing” and grammatical variants thereof as used herein refer to a nucleic acid editing technology that uses a Cas9 nickase fused to a reverse transcriptase and modifies a target nucleic acid without a double strand break or a donor DNA template. In Prime editing, the Cas9 nickase cuts the non-complementary strand of DNA upstream of the PAM site, thereby providing a 3’ flap that is extended with the extension including a modification. Further details on Prime editing can be found in Anzalone et al. (2019) Nature 576: 149-157 and / or U.S. Patent Application Publication No. 2021 / 0147862, the contents of each of which are incorporated herein by reference in their entirety.

[0087] In some embodiments, an editing system incorporates the Redraw editing system. Further details on the Redraw editing system can be found in U.S. Patent Application Publication No. 2021 / 0130835 and / or in U.S. Patent Application Publication No. 2022 / 0145334, the contents of each of which are incorporated herein by reference in their entirety.

[0088] As used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleotide sequence” and “polynucleotide” refer to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA / DNA hybrids. When dsRNA is produced synthetically, less common bases, such as inosine, 5-methylcytosine, 6- methyladenine, hypoxanthine and others can also be used for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides that contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression. Other modifications, such as modification to the phosphodiester backbone, or the 2'-hydroxy in the ribose sugar group of the RNA can also be made.

[0089] A “guide nucleic acid,” “guide RNA,” “gRNA,” “CRISPR RNA / DNA” “crRNA” or “crDNA” as used herein means a nucleic acid that comprises at least one spacer sequence, which is complementary to (and hybridizes to) a target nucleic acid e.g., a target DNA and / or a protospacer), and at least one repeat sequence (e.g., a repeat of a Type V Cast 2a CRISPR- Cas system, or a fragment or portion thereof; a repeat of a Type II Cas9 CRISPR-Cas system, or fragment thereof; a repeat of a Type V C2cl CRISPR Cas system, or a fragment thereof; a repeat of a CRISPR-Cas system of, for example, C2c3, Casl2a (also referred to as Cpfl), Cas 12b, Cas 12c, Cas 12d, Casl2e, Casl2f, Casl2i, Cas 13 a, Cas 13b, Cas 13c, Cas 13d, Casl, CaslB, Cas2, Cas3, Cas3’, Cas3”, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6,CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG), and / or Csf5, or a fragment thereof), wherein the repeat sequence may be linked to the 5’ end and / or the 3’ end of the spacer sequence. In some embodiments, the guide nucleic acid comprises DNA. In some embodiments, the guide nucleic acid comprises RNA (e.g., is a guide RNA). The design of a gRNA of this invention may be based on a Type I, Type II, Type III, Type IV, Type V, or Type VI CRISPR-Cas system.

[0090] In some embodiments, a Casl2a gRNA may comprise, from 5’ to 3’, a repeat sequence (full length or portion thereof (“handle”); e.g., pseudoknot-like structure) and a spacer sequence.

[0091] In some embodiments, a guide nucleic acid may comprise more than one repeat sequence-spacer sequence (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more repeat-spacer sequences) e.g., repeat-spacer-repeat, e.g., repeat-spacer-repeat-spacer-repeat-spacer-repeat-spacer-repeat- spacer, and the like). The guide nucleic acids of this invention are synthetic, human-made and not found in nature. A gRNA can be quite long and may be used as an aptamer (like in the MS2 recruitment strategy) or other RNA structures hanging off the spacer.

[0092] A “repeat sequence” as used herein, refers to, for example, any repeat sequence of a wild-type CRISPR Cas locus (e.g., a Cas9 locus, a Casl2a locus, a C2cl locus, etc.) or a repeat sequence of a synthetic crRNA that is functional with the CRISPR-Cas effector protein encoded by the nucleic acid constructs of the invention. A repeat sequence useful with this invention can be any known or later identified repeat sequence of a CRISPR-Cas locus (e.g., Type I, Type II, Type III, Type IV, Type V or Type VI) or it can be a synthetic repeat designed to function in a Type I, II, III, IV, V or VI CRISPR-Cas system. A repeat sequence may comprise a hairpin structure and / or a stem loop structure. In some embodiments, a repeat sequence may form a pseudoknot-like structure at its 5’ end (i.e., “handle”). Thus, in some embodiments, a repeat sequence can be identical to or substantially identical to a repeat sequence from wild-type Type I CRISPR-Cas loci, Type II, CRISPR-Cas loci, Type III, CRISPR-Cas loci, Type IV CRISPR-Cas loci, Type V CRISPR-Cas loci and / or Type VI CRISPR-Cas loci. A repeat sequence from a wild-type CRISPR-Cas locus may be determined through established algorithms, such as using the CRISPRfmder offered through CRISPRdb (see, Grissa et al. (2007) Nucl. Acids Res. 35(Web Server issue):W52-7). In some embodiments, a repeat sequence or portion thereof is linked at its 3’ end to the 5’ end of a spacer sequence, thereby forming a repeat-spacer sequence e.g., guide nucleic acid, guide RNA / DNA, crRNA, crDNA).

[0093] In some embodiments, a repeat sequence comprises, consists essentially of, or consists of at least 10 nucleotides depending on the particular repeat and whether the guide nucleic acid comprising the repeat is processed or unprocessed (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 to 100 or more nucleotides, or any range or value therein; e.g., about). In some embodiments, a repeat sequence comprises, consists essentially of, or consists of about 10 to about 20, about 10 to about 30, about 10 to about 45, about 10 to about 50, about 15 to about 30, about 15 to about 40, about 15 to about 45, about 15 to about 50, about 20 to about 30, about 20 to about 40, about 20 to about 50, about 30 to about 40, about 40 to about 80, about 50 to about 100 or more nucleotides.

[0094] A repeat sequence linked to the 5’ end of a spacer sequence can comprise a portion of a repeat sequence (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more contiguous nucleotides of a wild-type repeat sequence). In some embodiments, a portion of a repeat sequence linked to the 5’ end of a spacer sequence can be about five to about ten consecutive nucleotides in length (e.g., about 5, 6, 7, 8, 9, 10 nucleotides) and have at least 90% sequence identity (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the same region (e.g., 5’ end) of a wild-type CRISPR Cas repeat nucleotide sequence. In some embodiments, a portion of a repeat sequence may comprise a pseudoknot-like structure at its 5’ end (e.g., “handle”).

[0095] A “spacer sequence” as used herein is a nucleotide sequence that is complementary to a target nucleic acid (e.g., target DNA) (e.g., protospacer). The spacer sequence can be fully complementary or substantially complementary (e.g., at least about 70% complementary (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)) to a target nucleic acid. Thus, in some embodiments, the spacer sequence can have one, two, three, four, or five mismatches as compared to the target nucleic acid, which mismatches can be contiguous or noncontiguous. In some embodiments, the spacer sequence can have 70% complementarity to a target nucleic acid. In other embodiments, the spacer nucleotide sequence can have 80% complementarity to a target nucleic acid. In still other embodiments, the spacer nucleotide sequence can have 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% complementarity, and the like, to the target nucleic acid (protospacer). In some embodiments, the spacer sequence is 100% complementary to the target nucleic acid. A spacer sequence may have a length from about 15 nucleotides to about 30 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or any range or value therein). Thus, insome embodiments, a spacer sequence may have complete complementarity or substantial complementarity over a region of a target nucleic acid (e.g., protospacer) that is at least about 15 nucleotides to about 30 nucleotides in length. In some embodiments, the spacer is about 20 nucleotides in length. In some embodiments, the spacer is about 21, 22, or 23 nucleotides in length.

[0096] In some embodiments, the 5’ region of a spacer sequence of a guide nucleic acid may be fully complementary to a target nucleic acid, while the 3’ region of the spacer may be substantially complementary to the target nucleic acid (such as for a spacer in a Type V CRISPR-Cas system), or the 3’ region of a spacer sequence of a guide nucleic acid may be fully complementary to a target nucleic acid, while the 5’ region of the spacer may be substantially complementary to the target nucleic acid (such as for a spacer in a Type II CRISPR-Cas system), and therefore, the overall complementarity of the spacer sequence to the target nucleic acid may be less than 100%. Thus, for example, in a guide nucleic acid for a Type V CRISPR-Cas system, the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides in the 5’ region (z.e., seed region) of, for example, a 20 nucleotide spacer sequence may be 100% complementary to the target nucleic acid, while the remaining nucleotides in the 3’ region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target nucleic acid. In some embodiments, the first 1 to 8 nucleotides (e.g., the first 1, 2, 3, 4, 5, 6, 7, 8, nucleotides, and any range therein) of the 5’ end of the spacer sequence may be 100% complementary to the target nucleic acid, while the remaining nucleotides in the 3’ region of the spacer sequence are substantially complementary (e.g., at least about 50% complementary (e.g., 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)) to the target nucleic acid.

[0097] As a further example, in a guide nucleic acid for a Type II CRISPR-Cas system, the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides in the 3’ region (i.e., seed region) of, for example, a 20 nucleotide spacer sequence may be 100% complementary to the target nucleic acid, while the remaining nucleotides in the 5’ region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target nucleic acid. In some embodiments, the first 1 to 10 nucleotides (e.g., the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides, and any range therein) of the 3’ end of the spacer sequence may be 100% complementary to the target nucleic acid, while the remaining nucleotides in the 5’ region of the spacer sequence are substantially complementary (e.g., at least about 50% complementary (e.g., at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more or any range or value therein)) to the target nucleic acid. A recruiting guide RNA further comprises one or more recruiting motifs as described herein, which may be linked to the 5' end of the guide or the 3' end or it may be inserted into the recruiting guide nucleic acid (e.g., within the hairpin loop).

[0098] A “recruiting motif’ as used herein refers to one half of a binding pair that may be used to recruit a compound to which the recruiting motif is bound to another compound that includes the other half of the binding pair (z.e., a “corresponding motif’). The recruiting motif and corresponding motif may bind covalently and / or noncovalently. In some embodiments, a recruiting motif is an RNA recruiting motif (e.g., an RNA recruiting motif that is capable of binding and / or configured to bind to an affinity polypeptide), an affinity polypeptide (e.g., an affinity polypeptide that is capable of binding and / or configured to bind an RNA recruiting motif and / or a peptide tag), or a peptide tag (e.g., a peptide tag that is capable of binding and / or configured to bind an affinity polypeptide). For example, when a recruiting motif is an RNA recruiting motif, the corresponding motif for the RNA recruiting motif may be an affinity polypeptide that binds the RNA recruiting motif. A further example is that when a recruiting motif is a peptide tag, the corresponding motif for the peptide tag may be an affinity polypeptide that binds the peptide tag. Thus, a compound comprising a recruiting motif (e.g., an affinity polypeptide) may be recruited to another compound (e.g., a guide nucleic acid) comprising a corresponding motif for the recruiting motif (e.g., an RNA recruiting motif). Exemplary peptide tags (e.g., epitope) include, but are not limited to, a GCN4 peptide tag (e.g., Sun-Tag), a c-Myc affinity tag, an HA affinity tag, a His affinity tag, an S affinity tag, a methionine-His affinity tag, an RGD-His affinity tag, a FLAG® octapeptide, a strep tag or strep tag II, a V5 tag, and / or a VSV-G epitope.

[0099] In some embodiments, a seed region of a spacer may be about 8 to about 10 nucleotides in length, about 5 to about 6 nucleotides in length, or about 6 nucleotides in length.

[0100] A “target nucleic acid”, “target DNA,” “target nucleotide sequence,” “target region,” and “target region in the genome” are used interchangeably herein and refer to a region of an organism’s (e.g., a plant’s) genome that comprises a sequence that is fully complementary (100% complementary) or substantially complementary (e.g., at least 70% complementary (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)) to a spacer sequence in a guide nucleic acid as defined herein. A target nucleic acid is targeted by an editing system (or a component thereof) as described herein. A target regionuseful for a CRISPR-Cas system may be located immediately 3’ (e.g., Type V CRISPR-Cas system) or immediately 5’ (e.g., Type II CRISPR-Cas system) to a PAM sequence in the genome of the organism (e.g., a plant genome). A target region may be selected from any region of at least 15 consecutive nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides, and the like) located immediately adjacent to a PAM sequence. A target nucleic acid may be in a gene for which it is desired to alter the expression and / or activity of the product encoded thereby (e.g., protein and / or RNA).

[0101] A “protospacer sequence” or “protospacer” as used herein refer to a sequence that is fully or substantially complementary to (and can hybridize to) a spacer sequence of a guide nucleic acid. In some embodiments, the protospacer is all or a portion of a target nucleic acid as defined herein that is fully or substantially complementary (and hybridizes) to the spacer sequence of the CRISPR repeat-spacer sequences (e.g., guide nucleic acids, CRISPR arrays, crRNAs).

[0102] In the case of Type V CRISPR-Cas (e.g., Casl2a) systems and Type II CRISPR- Cas (Cas9) systems, the protospacer sequence is flanked by (e.g., immediately adjacent to) a protospacer adjacent motif (PAM). For Type IV CRISPR-Cas systems, the PAM is located at the 5’ end on the non-target strand and at the 3’ end of the target strand (see below, as an example).rand

[0103] In the case of Type II CRISPR-Cas (e.g., Cas9) systems, the PAM is located immediately 3’ of the target region. The PAM for Type I CRISPR-Cas systems is located 5’ of the target strand. There is no known PAM for Type III CRISPR-Cas systems. Makarova et al. describes the nomenclature for all the classes, types and subtypes of CRISPR systems (Makarova et al. (2015) Nature Rev. Microbiol. 13:722-736). Guide structures and PAMs are described by R. Barrangou ((2015) Genome Biol. 16:247).

[0104] Canonical Casl2a PAMs are T rich. In some embodiments, a canonical Casl2a PAM sequence may be 5’-TTN, 5’-TTTN, or 5’-TTTV. In some embodiments, canonical Cas9 (e.g., S. pyogenes) PAMs may be 5’-NGG-3’. In some embodiments, non-canonical PAMs may be used but may be less efficient.

[0105] Additional PAM sequences may be determined by those skilled in the art through established experimental and computational approaches. Thus, for example, experimentalapproaches include targeting a sequence flanked by all possible nucleotide sequences and identifying sequence members that do not undergo targeting, such as through the transformation of target plasmid DNA (Esvelt et al. (2013) Nat. Methods 10: 1116-1121; Jiang et al. (2013) Nat. Biotechnol. 31 :233-239). In some aspects, a computational approach can include performing BLAST searches of natural spacers to identify the original target DNA sequences in bacteriophages or plasmids and aligning these sequences to determine conserved sequences adjacent to the target sequence (Briner & Barrangou (2014) AppL Environ. Microbiol. 80:994-1001; Mojica et al. (2009) Microbiology 155:733-740).

[0106] In some embodiments, a guide nucleic acid further comprises a reverse transcriptase template and may be referred to as an extended guide nucleic acid. An “extended guide nucleic acid” as used herein is a guide nucleic acid as described herein that further comprises a reverse transcriptase template (RTT) and / or a primer binding site (PBS). In some embodiments, an extended guide nucleic acid is an engineered prime editing guide RNA (pegRNA). An extended guide nucleic acid may be a targeted allele guide RNA (tagRNA) or a stabilized targeted allele guide RNA (stagRNA). A “tagRNA” as used herein refers to an extended guide nucleic acid that comprises a PBS and a RTT and has target strand complementarity. A “stagRNA” as used herein refers to a tagRNA that comprises a stabilization motif. A stabilization motif may be present at the 3’ and / or 5’ end of a tagRNA. In some embodiments, a stabilization motif is present at the 3’ end of a tagRNA. Exemplary stabilization motifs include, but are not limited to, recruiting motifs, RNA hairpins, pseudoknot sequences, and / or PP7 motifs (e.g., a PP7 RNA hairpin sequence). In some embodiments, a stagRNA is a tagRNA that comprises a PP7 RNA hairpin sequence. In some embodiments, a CRISPR-Cas effector protein (e.g., a Type II or Type V CRISPR-Cas effector protein), a reverse transcriptase, and an extended guide nucleic acid can form a complex or are comprised in a complex.

[0107] In some embodiments, an extended guide nucleic acid comprises an extended portion that includes a primer binding site and a reverse transcriptase template, wherein the reverse transcriptase template comprises the modification (e.g., edit) to be incorporated into a target nucleic acid. In some embodiments, an extended guide nucleic acid comprises, at its 3' end, a primer binding site and a modification (e.g., an edit) to be incorporated into the target nucleic acid (e.g., a reverse transcriptase template). In some embodiments, an extended guide nucleic acid comprises: (1) a sequence that interacts (e.g., recruits and / or binds) with a CRISPR-Cas effector protein (e.g., a CRISPR-Cas nuclease), (2) a spacer having substantial complementary to a first site on a target nucleic acid (e.g., a CRISPR RNA (crRNA) (a firstcrRNA) and / or tracrRNA+crRNA (sgRNA)), and (3) a nucleic acid encoded repair template (e.g., an RNA encoded repair template) comprising a primer binding site and an RNA template (e.g., that encodes the modification to be incorporated into the target nucleic acid). In some embodiments, an extended guide nucleic acid (e.g., an extended guide RNA) may comprise, 5'-3', a spacer sequence, a repeat sequence, and an extended portion, the extended portion comprising, 5' to 3', a reverse transcriptase template and a primer binding site. In some embodiments, an extended guide nucleic acid may comprise, 5'-3', a spacer sequence, a repeat sequence and an extended portion, the extended portion comprising, 5' to 3', a primer binding site and a reverse transcriptase template. In some embodiments, an extended guide nucleic acid may comprise, 5'-3', an extended portion, a spacer sequence, and a repeat sequence, wherein the extended portion comprises, 5' to 3', a reverse transcriptase template and a primer binding site. In some embodiments, an extended guide nucleic acid may comprise, 5'-3', an extended portion, a spacer sequence, and a repeat sequence, wherein the extended portion comprises, 5' to 3', a primer binding site and a reverse transcriptase template.

[0108] According to some embodiments, an extended guide nucleic acid (e.g., a pegRNA) may have a structure and / or be designed as described in Anzalone et al. (2019) Nature 576(7785): 149-157. In some embodiments, an extended guide nucleic acid comprises a primer binding site (PBS) optionally having a sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides and a reverse transcriptase template (RT template) sequence optionally having a sequence of 65 nucleotides or more. In some embodiments, a PBS of an extended guide nucleic acid has a sequence of less than 15 nucleotides and has a sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides (e.g., a sequence of 5 or 6 nucleotides in length). The RT template sequence may be after the PBS sequence in the 5' to 3' direction. In some embodiments, the RT template sequence of the extended guide nucleic acid has a length of greater than 65 nucleotides and may comprise about 50 or more nucleotides of heterology relative to the target site (e.g., target nucleic acid), followed by about 15 or more nucleotides of homology relative to the target site. In some embodiments, the RT template sequence of the extended guide nucleic acid is after the PBS sequence and the RT template sequence has a length of greater than 65 nucleotides with the sequence including more than 50 nucleotides of heterology relative to the target site, followed by more than 15 nucleotides of homology relative to the target site. Accordingly, in some embodiments, when the extended guide nucleic acid is reverse transcribed, the resulting newly transcribed sequence may hybridize and / or is configured to hybridize with the unnicked strand of the target site, which may thereby create a heteroduplex DNA with a large insertion into the newly synthesized strand. Upon repair of thismismatched DNA, the resultant repaired DNA may contain a large insertion (e.g., greater than 50 nucleotides) of DNA sequence. In some embodiments, the method may provide a large deletion (e.g., greater than 50 nucleotides) of DNA sequence. In some embodiments, the PBS and the 15 or more nucleotides of homology to the target site may comprise homology arms, which may serve to insert the heterology into the target site optionally using homology directed repair. The inserted DNA may correspond to any functional sequence of DNA such as, but not limited to: a functional transgene; a fragment of DNA that is inserted into a gene in a way that, when the gene is transcribed, would produce a hairpin RNA that is sufficient to silence homologous genes through RNAi; and / or one or more functional site-specific recombination sites, e.g., lox, frt, which could then be used in subsequent Cre or Flp mediated site-specific recombination processes. In some embodiments, an extended guide nucleic acid may be too large to produce using a PolIII promoter in vivo. In some embodiments, an extended guide nucleic acid may be operatively associated with and / or produced using a PolII promoter. In some embodiments, a DNA binding polypeptide (e.g., a DNA binding domain) and / or DNA endonuclease may have a structure and / or be designed as described in Anzalone et al. (2019) Nature 576(7785): 149-157. In some embodiments, a DNA binding domain and / or DNA endonuclease is a CRISPR Cas polypeptide such as a Cas9 nickase, a nicking variant of another CRISPR Cas polypeptide, or Cas 12a.

[0109] In some embodiments, two extended guide nucleic acids (e.g., pegRNAs) may be used (e.g., an editing system may comprise two extended guide nucleic acids). One or both of the two extended guide nucleic acids may have a structure and / or be designed as described in Anzalone et al. (2019) Nature 576(7785): 149-157. The two extended guide nucleic acids may comprise a primer binding site (PBS) optionally having a sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides and a reverse transcriptase template (RT template) sequence optionally having a sequence of 50 nucleotides or more. The RT template sequences of the two extended guide nucleic acids may be complementary to each other and as such the polynucleotides that are respectively reverse transcribed from each the RT templates will be complementary to each other and will be able to hybridize with each other. This may allow for the intermediates that are produced by this system and / or method to join together two sections of DNA that are otherwise separated by more than 50 nucleotides, e.g., within a chromosome, or that are positioned on two separate pieces of DNA, e.g., on two different chromosomes. After repair of the intermediates, the resultant products may produce, depending on the design of the RT template, large deletions, large inversions, or inter-chromosomal recombinations. Since all of these products are produced by homology directed repair, the products may bepredictably precise and / or reproducible. In some embodiments, a DNA binding polypeptide e.g., a DNA binding domain) and / or DNA endonuclease may have a structure and / or be designed as described in Anzalone et al. (2019) Nature 576(7785): 149-157. In some embodiment, a DNA binding polypeptide and / or DNA endonuclease is a CRISPR Cas polypeptide such as a Cas9 nickase, a similar nicking variant of another CRISPR Cas polypeptide, or Casl2a. In some embodiments, a DNA binding polypeptide and / or DNA endonuclease is a Cas9 nuclease, a similar nuclease from another CRISPR Cas polypeptide, or Casl2a. Using a nuclease (rather than a nickase) may facilitate the intra- or interchromosomal recombination processes through single-strand annealing of the more than 50 nucleotide 3’ overhangs that would be produced at each of the two target sites corresponding to the two pegRNA target nucleic acids. In some embodiments, an editing system comprises one extended guide nucleic acid and a guide nucleic acid that is devoid of a reverse transcriptase template and / or primer binding site.

[0110] An extended guide nucleic acid may comprise a CRISPR nucleic acid (e.g., CRISPR RNA, CRISPR DNA, crRNA, crDNA) and / or a CRISPR nucleic acid and a tracr nucleic acid; and (b) an extended portion comprising a primer binding site and a reverse transcriptase template (RT template), wherein the RT template encodes a modification to be incorporated into the target nucleic acid. The CRISPR nucleic acid may be a Type II or TypeV CRISPR nucleic acid and / or the tracr nucleic acid may be any tracr corresponding to the appropriate Type II or Type V CRISPR nucleic acid. In some embodiments, an extended guide nucleic acid comprises: (i) a Type V CRISPR nucleic acid or a Type II CRISPR nucleic acid (e.g., a Type II or Type V CRISPR RNA, Type II or Type V CRISPR DNA, Type II or TypeV crRNA, or Type II or Type V crDNA) and / or a CRISPR nucleic acid and a tracr nucleic acid (e.g., a Type II or Type V tracrRNA, Type II or Type V tracrDNA); and (ii) an extended portion comprising a primer binding site and a reverse transcriptase template (RT template), wherein the Type V CRISPR nucleic acid or Type II CRISPR nucleic acid comprises a spacer that binds to a first strand (e.g., the target strand) of a target nucleic acid (e.g., the spacer is complementary to a portion of consecutive nucleotides in the first strand of the target nucleic acid) and the primer binding site binds to the first strand (e.g., target strand). In some embodiments, the extended portion can be fused to either the 5' end or 3' end of the CRISPR nucleic acid (e.g., from 5' to 3': repeat-spacer-extended portion or extended portion-repeat-spacer) and / or to the 5' or 3' end of the tracr nucleic acid. In some embodiments, the extended portion of an extended guide nucleic acid comprises, 5' to 3', an RT template (RTT) and a primer binding site (PBS) (e.g., 5’-crRNA-spacer-RTT(edit encoded)-PBS-3’) or comprises 5' to 3' a PBS and RTT,depending on the location of the extended portion relative to the CRISPR nucleic acid of the extended guide nucleic acid (e.g., 5’-crRNA-spacer-PBS-RTT(edit encoded)-3’). For example, in some embodiments, an extended portion of the extended guide nucleic acid may comprise, 5' to 3', an RT template and a primer binding site (when the extended guide is linked to the 3' end of the CRISPR nucleic acid). In some embodiments, an extended portion of the extended guide may comprise, 5' to 3', a primer binding site and an RT template (when the extended guide is linked to the 5' end of the CRISPR nucleic acid).

[0111] In some embodiments, a target nucleic acid is double stranded and comprises a first strand and a second strand and a primer binding site of an extended guide nucleic acid binds to the second strand (e.g., the non-target, top strand) of the target nucleic acid. In some embodiments, a target nucleic acid is double stranded and comprises a first strand and a second strand and a primer binding site of an extended guide nucleic acid binds to the first strand (e.g., binds to the target strand, optionally the same strand to which a CRISPR-Cas effector protein is recruited, bottom strand) of the target nucleic acid. In some embodiments, a target nucleic acid is double stranded and comprises a first strand and a second strand and the primer binding site of an extended guide nucleic acid binds to the second strand (e.g., the non-target strand, optionally the opposite strand from that to which the CRISPR-Cas effector protein is recruited) of the target nucleic acid. In some embodiments, a reverse transcriptase (RT) may add to the target strand of a target nucleic acid (e.g., the strand to which the spacer of the CRISPR nucleic acid of the extended guide nucleic acid is complementary and to which the CRISPR-Cas effector protein is recruited). In some embodiments, the reverse transcriptase (RT) adds to the non-target strand of a target nucleic acid (e.g., the strand that is complementary to the strand to which the spacer of the CRISPR nucleic acid is complementary and to which the CRISPR- Cas effector protein is recruited). Example methods and editing systems are described in International Patent Publication No. WO 2021 / 092130, International Patent Publication No. WO 2022 / 098993, and U.S. Patent Application Publication Nos. 2021 / 0147862, 2021 / 0130835, 2021 / 0147862, and 2022 / 0145334, each of which are incorporated herein by reference in their entirety.

[0112] The RT template of an extended guide nucleic acid may encode one or more modification(s) (e.g., edit(s)) to be incorporated into a target nucleic acid. The one or more modification(s) may be located in any position within an RT template (e.g., where the position location may be relative to the position of a protospacer adjacent motif (PAM) of the target nucleic acid). In some embodiments, an RT template has a modification at one or more positions from -1 to 23 (e.g., -1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, or 23) relative to the position of a protospacer adjacent motif (PAM) (e.g., TTTG) in a target nucleic acid. In some embodiments, an RT template may comprise a modification located at nucleotide position -1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23. In some embodiments, an RT template may comprise a modification located at nucleotide position 4 to nucleotide position 17 (e.g., position 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) of the RT template relative to the position of a PAM of a target nucleic acid. In some embodiments, an RT template may comprise a modification located at nucleotide position 10 to nucleotide position 17 (e.g., position 10, 11, 12, 13, 14, 15, 16, or 17) of the RT template relative to the position of a PAM of a target nucleic acid. In some embodiments, an RT template may comprise a modification located at nucleotide position 12 to nucleotide position 15 (e.g., position 12, 13, 14, or 15) of the RT template relative to the position of a PAM of a target nucleic acid.

[0113] In some embodiments, an extended portion of an extended guide nucleic acid may comprise, 5' to 3', an RT template and a primer binding site (e.g., when the extended portion is linked to the 3' end of a CRISPR nucleic acid). In some embodiments, an extended portion of an extended guide nucleic acid may comprise, 5' to 3', a primer binding site and an RT template (RTT) (e.g., when the extended portion is linked to the 5' end of the CRISPR nucleic acid). In some embodiments, an RT template may have a length of about 1 nucleotide to about 100 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100 or more nucleotides, and any range or value therein), e.g., about 1 nucleotide to about 10 nucleotides, about 1 nucleotide to about 15 nucleotides, about 1 nucleotide to about 20 nucleotides, about 1 nucleotide to about 25 nucleotides, about 1 nucleotide to about 30 nucleotides, about 1 nucleotide to about 35, 36, 37, 38, 39 or 40 nucleotides, about 1 nucleotide to about 50 nucleotides, about 5 nucleotides to about 15 nucleotides, about 5 nucleotides to about 20 nucleotides, about 5 nucleotides to about 25 nucleotides, about 5 nucleotides to about 30 nucleotides, about 5 nucleotides to about 35, 36, 37, 38, 39 or 40 nucleotides, about 5 nucleotides to about 50 nucleotides, about 8 nucleotides to about 15 nucleotides, about 8 nucleotide to about 20 nucleotides, about 8 nucleotide to about 25 nucleotides, about 8 nucleotide to about 30 nucleotides, about 8 nucleotide to about 35, 36, 37, 38, 39 or 40 nucleotides, about 8 nucleotide to about 50 nucleotides in length, about 8 nucleotides to about 100 nucleotides, about 10 nucleotide to about 15 nucleotides, about 10 nucleotide to about 20nucleotides, about 10 nucleotide to about 25 nucleotides, about 10 nucleotide to about 30 nucleotides, about 10 nucleotide to about 36 nucleotides, about 10 nucleotide to about 40 nucleotides, about 10 nucleotide to about 50 nucleotides, about 10 nucleotides to about 100 nucleotides in length and any range or value therein. In some embodiments, the length of an RT template may be at least 8 nucleotides, optionally about 8 nucleotides to about 100 nucleotides. In some embodiments, the length of an RT template is 36, 37, 38, 39 or 40 nucleotides or less (e.g, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length, or any value or range therein (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length to about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length). In some embodiments, the length of an RT template may be at least 30 nucleotides, optionally about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length to about to about 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides in length, or any range or value therein. In some embodiments, the length of an RT template may be about 36, 40, 44, 47, 50, 52, 55, 63, 72 or 74 nucleotides. Within the length of the RTT one or more modification(s) may be present. The one or more modification(s) may be located anywhere within the RTT, wherein the position of the modification may be described relative to the position of a protospacer adjacent motif (PAM) of a target nucleic acid. In some embodiments, an RT template may comprise a modification located at nucleotide position -1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23. In some embodiments, an RT template may comprise a modification located at nucleotide position 4 to nucleotide position 17 (e.g., position 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) of the RT template relative to the position of a protospacer adjacent motif (PAM) of a target nucleic acid. In some embodiments, an RT template may comprise a modification located at nucleotide position 10 to nucleotide position 17 (e.g., position 10, 11, 12, 13, 14, 15, 16, or 17) of the RT template relative to the position of a protospacer adjacent motif (PAM) of a target nucleic acid. In some embodiments, an RT template may comprise a modification located at nucleotide position 12 to nucleotide position 15 (e.g., position 12, 13, 14, or 15) of the RT template relative to the position of a protospacer adjacent motif (PAM) of a target nucleic acid.

[0114] As used herein, a “primer binding site” (PBS) of an extended portion of an extended guide nucleic acid (e.g., a tagRNA) refers to a sequence of consecutive nucleotides that can bind to a region or “primer” on a target nucleic acid, e.g., is complementary to the target nucleicacid primer. As an example, a CRISPR Cas effector protein (e.g., a Type II or Type V, e.g., Cas 9 or Casl2a) may nick / cut the DNA and the 3' end of the cut DNA acts as a primer for the PBS portion of the extended guide nucleic acid. The PBS may be complementary to the 3' end of a strand of the target nucleic acid and may bind and / or may be configured to bind to either the target strand or non-target strand. A primer binding site can be fully complementary to the primer or it may be substantially complementary (e.g., at least 70% complementary (e.g., 70% or about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more)) to the primer of a target nucleic acid. In some embodiments, the length of a primer binding site of an extended portion may be about 1 nucleotide to about 100 nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more nucleotides, or any value or range therein), or about 4 nucleotide to about 85 nucleotides, about 10 nucleotide to about 80 nucleotides, about 20 nucleotide to about 80 nucleotides, about 25 nucleotides to about 80 nucleotides about 30 nucleotide to about 80 nucleotides, about 40 nucleotide to about 80 nucleotides, about 45 nucleotide to about 80 nucleotides, about 45 nucleotide to about 75 nucleotides, or about 45 nucleotide to about 60 nucleotides, or any range or value therein. In some embodiments, the length of a PBS may be at least 30 nucleotides, optionally about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides to about 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides in length, or any range or value therein. In some embodiments, the length of a PBS may be about 8, 16, 24, 32, 40, 48, 56, 64, 72, or 80 nucleotides.

[0115] In some embodiments, an RTT may have a length of about 35 nucleotides to about 75 nucleotides and a PBS may have a length of about 30 nucleotides to about 80 nucleotides, optionally wherein the PBS may comprise a length of about 8, 16, 24, 32, 40, 48, 56, 64, 72, or 80 nucleotides and the RTT may comprise a length of about 36, 40, 44, 47, 50, 52, 55, 63, 72 or 74 nucleotides, or any combination thereof of the RTT length and / or PBS length.

[0116] In some embodiments, an extended portion of an extended guide nucleic acid may be fused to either the 5' end or 3' end of a Type II or a Type V CRISPR nucleic acid (e.g., 5' to 3': repeat-spacer-extended portion, or extended portion-repeat-spacer) and / or to the 5' or 3' end of the tracr nucleic acid. In some embodiments, when an extended portion is located 5' of thecrRNA, a Type V CRISPR-Cas effector protein is modified to reduce (or eliminate) selfprocessing RNAse activity.

[0117] In some embodiments, the extended portion of an extended guide nucleic acid may be linked to the Type II or Type V CRISPR nucleic acid and / or the Type II or Type V tracrRNA via a linker. In some embodiments, a linker have a length of about 1 to about 100 nucleotides or more (e.g, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100 or more nucleotides in length, and any range therein (e.g., about 2 to about 40, about 2 to about 50, about 2 to about 60, about 4 to about 40, about 4 to about 50, about 4 to about 60, about 5 to about 40, about 5 to about 50, about 5 to about 60, about 9 to about 40, about 9 to about 50, about 9 to about 60, about 10 to about 40, about 10 to about 50, about 10 to about 60, about 40 to about 100, about 50 to about 100, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides to about 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more nucleotides in length (e.g., about 105, 110, 115, 120, 130, 140 150 or more nucleotides in length).

[0118] A guide nucleic acid and / or an extended guide nucleic acid may comprise one or more recruiting motifs as described herein, which may be linked to the 5' end and / or the 3' end of the guide nucleic acid and / or it may be inserted into the guide nucleic acid (e.g., within a hairpin loop of the guide nucleic acid). In some embodiments, an extended guide nucleic acid may be linked to an RNA recruiting motif. An extended guide nucleic acid and / or guide nucleic acid may be linked to one or to two or more RNA recruiting motifs (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more motifs; e.g., at least 10 to about 25 motifs), optionally wherein the two or more RNA recruiting motifs may be the same RNA recruiting motif or different RNA recruiting motifs. In some embodiments, an RNA recruiting motif may be located on the 3' end of the extended portion of an extended guide nucleic acid (e.g., 5'-3', repeat-spacer-extended portion (RT template-primer binding site)-RNA recruiting motif). In some embodiments, an RNA recruiting motif may be embedded in the extended portion of an extended guide nucleic acid.

[0119] In some embodiments, an editing system comprises an extended guide nucleic acid that is linked to an RNA recruiting motif and a reverse transcriptase that is a reverse transcriptase fusion protein, wherein the reverse transcriptase fusion protein comprises areverse transcriptase polypeptide fused to an affinity polypeptide that binds to the RNA recruiting motif, wherein the extended guide nucleic acid binds to a target nucleic acid and the RNA recruiting motif binds to the affinity polypeptide, thereby recruiting the reverse transcriptase fusion protein to the extended guide nucleic acid and contacting the target nucleic acid with the reverse transcriptase. In some embodiments, two or more reverse transcriptase fusion proteins may be recruited to an extended guide nucleic acid, thereby contacting the target nucleic acid with two or more reverse transcriptase fusion proteins.

[0120] As used herein, a “CRISPR-Cas effector protein” is a protein or polypeptide that cleaves, cuts, or nicks a nucleic acid; binds a nucleic acid (e.g., a target nucleic acid and / or a guide nucleic acid); and / or that identifies, recognizes, or binds a guide nucleic acid as defined herein. In some embodiments, a CRISPR-Cas effector protein may be an enzyme (e.g., a nuclease, endonuclease, nickase, etc.) and / or may function as an enzyme. In some embodiments, a CRISPR-Cas effector protein refers to a CRISPR-Cas nuclease. In some embodiments, a CRISPR-Cas effector protein comprises nuclease activity and / or nickase activity, comprises a nuclease domain whose nuclease activity and / or nickase activity has been reduced or eliminated, comprises single stranded DNA cleavage activity (ss DNAse activity) or which has ss DNAse activity that has been reduced or eliminated, and / or comprises selfprocessing RNAse activity or which has self-processing RNAse activity that has been reduced or eliminated. A CRISPR-Cas effector protein may bind to a target nucleic acid. A CRISPR- Cas effector protein may be a Type I, II, III, IV, V, or VI CRISPR-Cas effector protein. In some embodiments, a CRISPR-Cas effector protein may be from a Type I CRISPR-Cas system, a Type II CRISPR-Cas system, a Type III CRISPR-Cas system, a Type IV CRISPR-Cas system, Type V CRISPR-Cas system, or a Type VI CRISPR-Cas system. In some embodiments, a CRISPR-Cas effector protein of the invention may be from a Type II CRISPR- Cas system or a Type V CRISPR-Cas system. In some embodiments, a CRISPR-Cas effector protein may be a Type II CRISPR-Cas effector protein, for example, a Cas9 effector protein. In some embodiments, a CRISPR-Cas effector protein may be Type V CRISPR-Cas effector protein, for example, a Cast 2 effector protein.

[0121] Exemplary CRISPR-Cas effector proteins include, but are not limited to, a Cas9, C2cl, C2c3, Cast 2a (also referred to as Cpfl), Cast 2b, Cast 2c, Cast 2d, Casl2e, Cast 3 a, Casl3b, Casl3c, Casl3d, Casl, CaslB, Cas2, Cas3, Cas3', Cas3”, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG),and / or Csf5 nuclease, optionally wherein the CRISPR-Cas effector protein may be a Cas9, Casl2a (Cpfl), Casl2b, Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2g, Casl2h, Casl2i, C2c4, C2c5, C2c8, C2c9, C2cl0, Casl4a, Casl4b, and / or Casl4c effector protein.

[0122] In some embodiments, a CRISPR-Cas effector protein useful with the invention may comprise a mutation in its nuclease active site and / or nuclease domain (e.g., a RuvC, HNH, e.g., a RuvC site of a Casl2a nuclease domain; e.g., a RuvC site and / or HNH site of a Cas9 nuclease domain). A CRISPR-Cas effector protein having a mutation in its nuclease active site and / or nuclease domain, and therefore, no longer comprising nuclease activity, is commonly referred to as “inactive” or “dead,” e.g., dCas9. In some embodiments, a CRISPR- Cas effector protein having a mutation in its nuclease active site and / or nuclease domain may have impaired activity or reduced activity (e.g., nickase activity) as compared to the same CRISPR-Cas effector protein without the mutation.

[0123] A CRISPR Cas9 effector protein or Cas9 useful with this invention may be any known or later identified Cas9 nuclease. In some embodiments, a Cas9 of the present invention may be a protein from, for example, Streptococcus spp. (e.g., S. pyogenes, S. thermophilus'), Lactobacillus spp., Bifidobacterium spp., Kandleria spp., Leuconostoc spp., O enococcus spp., Pediococcus spp., Weissella spp., and / or Olsenella spp.

[0124] In some embodiments, the CRISPR-Cas effector protein may be a Cas9 derived from Streptococcus pyogenes and / or may recognize the PAM sequence motif NGG, NAG, NGA (Mali et al. (2013) Science 339(6121):823-826). In some embodiments, the CRISPR-Cas effector protein may be a Cas9 derived from Streptococcus thermophilus and / or may recognize the PAM sequence motif NGGNG and / or NNAGAAW (W = A or T) (See, e.g., Horvath et al. (2010) Science 327(5962): 167-170; Deveau et al. (2008) J. Bacteriol. 190(4): 1390-1400). In some embodiments, the CRISPR-Cas effector protein may be a Cas9 derived from Streptococcus mutans and / or may recognize the PAM sequence motif NGG and / or NAAR (R = A or G) (See, e.g., Deveau et al. (2008) J. Bacteriol. 190(4): 1390-1400). In some embodiments, the CRISPR-Cas effector protein may be a Cas9 derived from Streptococcus aureus and / or may recognize the PAM sequence motif NNGRR (R = A or G). In some embodiments, the CRISPR-Cas effector protein may be a Cas9 derived from S. aureus and / or may recognize the PAM sequence motif NGRRT (R = A or G). In some embodiments, the CRISPR-Cas effector protein may be a Cas9 derived from S. aureus and / or may recognize the PAM sequence motif NGRRV (R = A or G). In some embodiments, the CRISPR-Cas effector protein may be a Cas9 that is derived from Neisseria meningitidis and / or may recognize the PAM sequence motif NGATT or NGCTT (R = A or G, V = A, G or C) (See, e.g., Hou et al.(2013) PNAS 110(35): 15644-15649). In the aforementioned embodiments in this paragraph, N in the PAM sequence motif can be any nucleotide residue, e.g., any of A, G, C or T. In some embodiments, the CRISPR-Cas effector protein may be a Cast 3a derived from Leptotrichia shahii and / or may recognize a protospacer flanking sequence (PFS) (or RNA PAM (rPAM)) sequence motif of a single 3’ A, U, or C, which may be located within the target nucleic acid.

[0125] A Type V CRISPR-Cas effector protein useful with embodiments of the invention may be any Type V CRISPR-Cas nuclease. Exemplary Type V CRISPR-Cas effector proteins include, but are not limited, to Casl2a (Cpfl), Casl2b, Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2g, Casl2h, Casl2i, C2cl, C2c4, C2c5, C2c8, C2c9, C2cl0, Casl4a, Casl4b, and / or Casl4c nuclease. In some embodiments, a Type V CRISPR-Cas effector protein may be a Cast 2a. In some embodiments, a Type V CRISPR-Cas effector protein may be a nickase, optionally, a Cast 2a nickase.

[0126] In some embodiments, the CRISPR-Cas effector protein may be a Type V Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas nuclease. Cast 2a differs in several respects from the more well-known Type II CRISPR Cas9 nuclease. For example, Cas9 recognizes a G-rich protospacer-adjacent motif (PAM) that is 3' to its guide RNA (gRNA, sgRNA, crRNA, crDNA, CRISPR array) binding site (protospacer, target nucleic acid, target DNA) (3'-NGG), while Casl2a recognizes a T-rich PAM that is located 5' to the target nucleic acid (5'-TTN, 5'-TTTN). In fact, the orientations in which Cas9 and Casl2a bind their guide RNAs are very nearly reversed in relation to their N and C termini. Furthermore, Cast 2a enzymes use a single guide RNA (gRNA, CRISPR array, crRNA) rather than the dual guide RNA (sgRNA (e.g., crRNA and tracrRNA) found in natural Cas9 systems, and Casl2a processes its own gRNAs. Additionally, Casl2a nuclease activity produces staggered DNA double stranded breaks instead of blunt ends produced by Cas9 nuclease activity, and Cast 2a relies on a single RuvC domain to cleave both DNA strands, whereas Cas9 utilizes an HNH domain and a RuvC domain for cleavage.

[0127] A CRISPR Casl2a effector protein useful with this invention may be any known or later identified Casl2a (previously known as Cpfl) (see, e.g., U.S. Patent No. 9,790,490, which is incorporated by reference for its disclosures of Cpfl (Casl2a) sequences). The term “Cast 2a” refers to an RNA-guided protein that can have nuclease activity, the protein comprising a guide nucleic acid binding domain and an active, inactive, or partially active DNA cleavage domain, thereby the RNA-guided nuclease activity of the Cast 2a may be active, inactive or partially active, respectively. In some embodiments, a Casl2a useful with the invention may comprise a mutation in the nuclease active site (e.g., a RuvC site of the Casl2adomain). A Casl2a having a mutation in its nuclease domain and / or nuclease active site, and therefore, no longer comprising nuclease activity, is commonly referred to as deadCasl2a (e.g., dCasl2a). In some embodiments, a Cast 2a having a mutation in its nuclease domain and / or nuclease active site may have impaired activity, e.g., may have reduced nickase activity.

[0128] In some embodiments, a CRISPR-Cas effector protein may be optimized for expression in an organism, for example, in an animal (e.g., a mammal such as a human), a plant, a fungus, an archaeon, or a bacterium. In some embodiments, a CRISPR-Cas effector protein (e.g., Casl2a polypeptide / domain or a Cas9 polypeptide / domain) may be optimized for expression in a plant.

[0129] In some embodiments, a CRISPR-Cas effector protein comprising a Cas9 domain (or a nucleic acid construct encoding the same) may be used in combination with a Cas9 guide nucleic acid to modify a target nucleic acid, and may be in or may form a complex.

[0130] Likewise, a CRISPR-Cas effector protein may comprise a Casl2a domain (or other selected CRISPR-Cas nuclease, e.g., C2cl, C2c3, Casl2b, Casl2c, Casl2d, Casl2f, Casl2i, Casl2e, Casl3a, Casl3b, Casl3c, Casl3d, Casl, CaslB, Cas2, Cas3, Cas3’, Cas3”, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG), and / or Csf5), which may be used in combination with a Casl2a guide nucleic acid (or the guide nucleic acid for the other selected CRISPR-Cas nuclease) to modify a target nucleic acid, thereby editing the target nucleic acid.

[0131] A method of the present invention may comprise producing a modified Rubus spp. plant or plant part from a transformed tissue produced according to embodiments of the present invention. In some embodiments, the modified Rubus spp. plant or plant part comprises a gene that confers resistance to a selection agent and / or polynucleotide of interest e.g., a polynucleotide encoding or comprising a portion or all of an editing system). In some embodiments, the modified Rubus spp. plant or plant part may be a transgenic plant or plant part. In some embodiments, the modified Rubus spp. plant or plant part plant may be non- transgenic and / or transgene free. In some embodiments, the modified Rubus spp. plant or plant part may be produced from a transgene-free shoot that comprises cells including a modified nucleic acid (e.g., edited gene), wherein the modified nucleic acid is provided and / or produced in accordance with a method of the present invention (e.g., as a result of introducing a polynucleotide of interest into a cell of wounded plant tissue as described herein). In some embodiments, a modified Rubus spp. plant or plant part is fully modified in that all cells of theplant or plant part include a modified nucleic acid. A transgene-free, edited Rubus spp. plant may be produced from a modified shoot comprising cells including the modified nucleic acid (e.g., an edited gene). In some embodiments, a modified Rubus spp. plant is produced from a modified shoot and / or modified root, and the modified Rubus spp. plant may comprise cells including a modified nucleic acid.

[0132] A method herein may comprise screening for a polynucleotide introduced by an Agrobacterium strain, a modified nucleic acid, and / or a given phenotype. In some embodiments, a transformed tissue, shoot or root is screened for a polynucleotide introduced by an Agrobacterium strain, a modified nucleic acid, and / or a given phenotype. In some embodiments, a progeny plant is screened for a polynucleotide introduced by an Agrobacterium strain, a modified nucleic acid, and / or a given phenotype. Screening may comprise phenotyping. In some embodiments, a method herein may comprise performing molecular screening such as molecular screening on a transformed tissue, root, shoot, and / or progeny plant. The molecular screening may be performed before and / or after phenotyping. Methods of screening are known to those of skill in the art and include, but are not limited to, evaluating gene expression levels such as by using quantitative PCR (qPCT) or sequencing and / or by physical and / or visual evaluation of the phenotype.

[0133] In some embodiments, a method and / or culturing step of the present invention may be used to produce a modified (e.g., edited) plant, plant part, and / or cell thereof. In some embodiments, the plant or plant part is from a species of Rubus. In some embodiments, the Rubus species is Rubus allegheniensis (Allegheny blackberry), Rubus armeniacus (Himalayan blackberry), Rubus canadensis (Canadian blackberry), Rubus fruticosus agg. (European blackberry), Rubus laciniatus (cutleaf evergreen blackberry), Rubus pensilvanicus (Pennsylvania blackberry), Rubus trifidus (Japanese blackberry), Rubus odoratus (purple- flowered raspberry) or Rubus ursinus (trailing blackberry).

[0134] The present invention further comprises a kit or kits to carry out the methods of this invention. A kit of this invention can comprise reagents, buffers, and apparatus for mixing, measuring, sorting, labeling, etc., as well as instructions and the like as would be appropriate for modifying a target nucleic acid.

[0135] In some embodiments, the invention provides a kit for comprising an Agrobacterium strain and / or one or more nucleic acid constructs of the invention, and / or expression cassettes and / or vectors and / or cells comprising the same as described herein, with optional instructions for the use thereof. In some embodiments, a kit may further comprise a CRISPR-Cas guide nucleic acid (corresponding to the CRISPR-Cas effector protein encodedby the polynucleotide of the invention) and / or expression cassettes and / or vectors and or cells comprising the same. In some embodiments, a guide nucleic acid may be provided on the same expression cassette and / or vector as one or more nucleic acid constructs of the invention. In some embodiments, the guide nucleic acid may be provided on a separate expression cassette or vector from that comprising the one or more nucleic acid constructs of the invention.

[0136] Accordingly, in some embodiments, kits are provided comprising an Agrobacterium strain and / or a nucleic acid construct comprising (a) a polynucleotide(s) as provided herein and (b) a promoter that drives expression of the polynucleotide(s) of (a). In some embodiments, the kit may further comprise a nucleic acid construct encoding a guide nucleic acid, wherein the construct comprises a cloning site for cloning of a nucleic acid sequence identical or complementary to a target nucleic acid sequence into backbone of the guide nucleic acid.

[0137] The invention will now be described with reference to the following examples. It should be appreciated that these examples are not intended to limit the scope of the claims to the invention, but are rather intended to be exemplary of certain embodiments. Any variations in the exemplified methods that occur to the skilled artisan are intended to fall within the scope of the invention.EXAMPLES

[0138] Example 1: Transformation Methods

[0139] Early attempts at generating Rubus transformants using leaf or petiole approaches described in the literature were performed with three black raspberry lines and seven blackberry lines (Table 1) across thousands of inoculated explants. Use of these conventional leaf or petiole approaches resulted in no transgenic shoots being generated. In direct contrast to reports in the literature, escape of wild-type (WT) material at hygromycin concentrations up to 30 mg / L was observed in both blackberry and black raspberry when using the leaf or petiole approaches. Thus, hygromycin was not a suitable selection agent for this type of approach.

[0140] Meristem -based transformation systems for Rubus spp. were subsequently developed focusing on the blackberry line BK-13. In the meristem-based transformation systems, WT escapes were observed in blackberry when using kanamycin at concentrations up to 75 mg / L. Accordingly, 100 mg / L kanamycin was used as the selection concentration for stable transformation, which is more than double any existing concentration used in the literature. Additionally, while some silencing of the visual marker, zsGreen, was observed, most of the transgenic plants produced clearly expressed this marker at a high level, asobserved by visual inspection, or included the coding sequence of this marker, as determined by PCR analysis.Table 1. Genotypes or species tested for leaf / petiole transformation.

[0141] Example 2: Proliferating Meristem tissues as explants and use for transformation

[0142] A system was developed for three different lines of Rubus to generate a transformable, proliferative culture. Briefly, nodal meristem tissues were introduced into tissue culture in the presence of moderate levels of TDZ (2 mg / L), low levels of IAA (0.01 mg / L), and a low level of hygromycin (5 mg / L). The proliferating early-stage shoot cultures were selectively excised and allowed to grow for no more than 12 weeks to generate clusters of proliferating meristematic tissue suitable for transformation. These clusters of proliferating meristems were transformed and either spectinomycin (150 mg / L) or kanamycin (100 mg / L) selection was used to select the transformed tissue. The selected transformed tissue was then cultured on a shoot proliferation medium followed by rooting medium, both of which included either spectinomycin (150 mg / L) or kanamycin (100 mg / L), to produce rooted transgenic tissue after three and six months from the date of transformation.

[0143] Example 3: Nodal meristem tissues as explants for biolistic-mediated transformation and stringent spectinomycin selection.

[0144] A system was developed for one line of Rubus to generate transgenic plants. Briefly, BK-28 nodal meristem explants were harvested from the greenhouse and introduced into culture. Shoot apical meristems contained on dormant buds were carefully excised to expose multiple underlying meristems within the same dormant bud. Intact buds on cane segments were then placed onto a basal medium and allowed to grow for 2-3 days in culture, so that exposed lateral meristems would emerge from the bud as target tissue for biolistic transformation. After 2-3 days, buds were carefully excised from the cane segment under a microscope and arranged 25 to the center of a petri dish with the newly emerged meristems facing upwards for particle bombardment. Emerged meristems were bombarded with a polynucleotide of interest using a PDS-1000 gene gun. One to two days after bombardment, explants were transferred to a cytokinin-containing medium that included spectinomycin at 150 mg / 1 for three weeks, and were then refreshed onto new medium for transgenic shoot emergence where the new medium could include an auxin and could be devoid of a growth regulator and / or a cytokinin. Once emerged, green transgenic shoots expressing a visual marker could then be moved onto basal medium containing 0.1 mg / 1 IBA and 50 mg / 1 spectinomycin for rooting. Regeneration of transgenic plants was achieved three to four months after transformation. The results are provided in Table 2.Table 2.

[0145] Example 4: Blender transformation method with stringent kanamycin selection or spectinomycin selection.

[0146] In vitro shoot clusters were generated using a propagation system either three or six weeks prior to use for transformation in order to generate a suitable amount of tissue for transformation. Whole shoots were blended in a disarmed Agrobacterium tumefaciens solution of 0.8 OD, and were sonicated, vacuumed, centrifuged, and plated out for culture withsubsequent regeneration under kanamycin (100 mg / L) selection or spectinomycin (150 mg / L). Initially, delivery and inoculation parameters were based upon transient zsGreen expression. Transgenic plants were generated by culturing on shoot proliferation medium and the root induction medium, both of which included kanamycin (100 mg / L) or spectinomycin (150 mg / L). Regeneration of plants was achieved through the proliferation of meristematic cells, under selective conditions three to four months after transformation. Because of the regenerable nature of meristematic cells, a wide range of growth regulators was suitable for this method (Table 3), and transgenic events were produced on multiple different media if selection levels were high (e.g., at least kanamycin (100 mg / L) or spectinomycin (150 mg / L)). The cytokinins BA (benzylaminopurine) or TDZ (thidiazuron) could be used alone or in combination with the auxin IBA (indole-3 -butyric acid) to recover transformed plants. Various wounding and inoculation methods were tested, to improve contact between Agrobacterium and regenerable cells within meristematic layers (Table 4). Although shoot clusters that had been cultured in vitro for 3 weeks were initially used to generate transformation explants, clusters that had undergone additional multiplication out to 6 weeks were also suitable to produce transformed events (Table 5) at similar or better transformation frequencies. Both high levels of kanamycin and spectinomycin could be used to generate transformed plants (Tables 6 and 7), and the stringency of selection was essential for recovery of transformed plants and eliminated untransformed “escape” plants from forming. As an alternative to using the selectable markers nptll and aadA within the T-DNA, a co-editing approach was taken to simultaneously edit marl and trait targets to obtain kanamycin-resistant edited plants. For this approach to be successful, kanamycin doses had to be lowered to 75 or 50 mg / 1 in order to recover edited events which resulted in a significant amount of escaped non-transgenic plants. Using this coediting approach, marl edited plants were recovered that contained up to twelve other additional edited alleles across three trait gene targets (Table 8).Table 3. Regeneration of transgenic plants from blender-transformed 3 week explants after inoculation with a disarmed A. tumefaciens, regenerated under 100 mg / L kanamycin selection on a wide range of media.Table 4. Regeneration of transgenic plants from blender-transformed 3 week explants after inoculation with a disarmed A. tumefaciens, regenerated under 100 mg / L kanamycin selection using multiple inoculation or delivery parameters.Table 5. Regeneration of transgenic plants from blender-transformed explants after inoculation with a disarmed A. tumefaciens, regenerated under 100 mg / L kanamycin selection using either 3 week or 6 week propagated explants.Table 6. Regeneration of transgenic plants from blender-transformed 3 week explants after inoculation with a disarmed A. tumefaciens, regenerated under 100 mg / L kanamycin selection across two constructs and using different growth regulators.Table 7. Regeneration of transgenic plants from blender-transformed 3 week explants after inoculation with a disarmed A. tumefaciens, regenerated under 150 mg / L spectinomycin selection across two constructs.Table 8. Regeneration of transgenic marl- and trait-edited plants from blender- transformed 3 week explants after inoculation with a disarmed A. tumefaciens, regenerated under two kanamycin concentrations.The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

WHAT IS CLAIMED IS:

1. A method of producing a modified Rubus species (Rubus spp.) plant or plant part, the method comprising: wounding a Rubus spp. tissue comprising meristem cells to provide a wounded tissue comprising the meristem cells; introducing a polynucleotide of interest and a gene that confers resistance to a selection agent into one or more meristem cells of the wounded tissue to provide a transformed tissue; and culturing the transformed tissue in the presence of the selection agent in an amount at least 1.5 times that needed to kill 90% of Rubus spp. cells to provide a cultured transformed tissue; and regenerating a modified Rubus spp. plant or plant part from the cultured transformed tissue.

2. A method of producing a modified Rubus species (Rubus spp.) plant or plant part, the method comprising: wounding a Rubus spp. tissue comprising meristem cells to provide a wounded tissue comprising the meristem cells; introducing at least a portion of an editing system and a gene that confers resistance to a selection agent into one or more meristem cells of the wounded tissue to provide a transformed tissue, wherein the portion of the editing system is targeted to a region of a gene of interest that is endogenous to the Rubus spp.; culturing the transformed tissue in the presence of the selection agent to provide a cultured transformed tissue, wherein the selection agent is in an amount at least 1.5 times that needed to kill 90% of Rubus spp. cells; and regenerating a modified Rubus spp. plant or plant part from the cultured transformed tissue.

3. The method of claim 2, further comprising introducing, via the editing system, a mutation into the gene of interest to provide an edited tissue.

4. The method of any one of the preceding claims, wherein the Rubus spp. tissue is a Rubus allegheniensis, Rubus occidenlalis. Rubus odoralus. o Rubus fruticosus tissue.

5. The method of any one of the preceding claims, wherein the Rubus spp. tissue is shoot tissue, optionally wherein the Rubus spp. tissue comprises meristem tissue and / or a node.

6. The method of any one of the preceding claims, wherein the selection agent is kanamycin and the kanamycin is present in an amount of at least about 75 mg / L, optionally wherein the kanamycin is present in an amount of at about 100 mg / L.

7. The method of any one of claims 1-5, wherein the selection agent is spectinomycin and the spectinomycin is present in an amount of at least about 150 mg / L.

8. The method of any one of the preceding claims, wherein prior to wounding the Rubus spp. tissue, the method comprises the step of culturing a Rubus spp. shoot material comprising a node in the presence of at least one plant growth regulator to produce the Rubus spp. tissue comprising meristem cells (e.g., shoot meristem cells).

9. The method of claim 8, wherein the at least one plant growth regulator comprises a cytokinin, a cytokinin-like compound, an auxin, an auxin precursor, and / or an antibiotic, optionally wherein the least one plant growth regulator comprises thidiazuron (TDZ) and hygromycin.

10. The method of claim 9, wherein the cytokinin or cytokinin-like compound is selected from thidiazuron (TDZ), 6-benzylaminopurine (BAP), kinetin, forchlorfenuron (4-CPPU), 6- (y -dimethylallylamino) purine (2iP), zeatin, zeatin-riboside, dihydro-zeatin, adenine, 6- isopentenyladenine (IP A), and / or TIBA (2,3,5-triiodobenzoic acid).

11. The method of any one of claims 8-10, wherein culturing the Rubus spp. shoot material comprising the node in the presence of the at least one growth regulator to produce xe. Rubus spp. tissue comprising meristem cells (e.g., shoot meristem cells) comprises culturing the Rubus spp. shoot material in the presence of thidiazuron (TDZ) in an amount of about 0.1 mg / L to about 5 mg / L and in the presence of hygromycin in an amount of about 1 mg / L to about 10 mg / L.

12. The method of any one of the preceding claims, wherein the Rubus spp. tissue comprising meristem cells (e.g., shoot meristem cells) comprises a proliferating meristem tissue and / or a proliferating shoot.

13. The method of any one of the preceding claims, wherein the transformed tissue and / or the cultured transformed tissue comprises a transgenic cell, optionally wherein the transformed tissue is a transformed shoot, optionally wherein the transgenic cell is an edited cell.

14. The method of any one of the preceding claims, wherein the transformed tissue and / or the cultured transformed tissue comprises a wild-type cell and a transgenic cell optionally wherein the transformed tissue is a transformed shoot.

15. The method of any one of the preceding claims, wherein the transformed tissue and / or the cultured transformed tissue comprises at least two different edited cells optionally wherein the transformed tissue is a transformed shoot.

16. The method of any one of the preceding claims, wherein the regenerating step comprises culturing the cultured transformed tissue on a proliferation medium to produce a shoot and subsequently culturing the shoots on root induction medium to provide the modified Rubus spp. plant or plant part.

17. The method of claim 16, wherein the modified Rubus spp. plant or plant part is mosaic.

18. The method of claim 16, wherein the modified Rubus spp. plant or plant part is nonmosaic.

19. The method of any one of the preceding claims, wherein introducing the polynucleotide of interest, the gene that confers resistance to the selection agent and / or the at least a portion of the editing system into the one or more meristem cells of the wounded tissue comprises contacting the wounded tissue with an Agrobacterium that comprises thepolynucleotide of interest, the gene that confers resistance to the selection agent and / or the at least a portion of the editing system.

20. The method of claim 19, wherein the wounding and contacting steps are carried out simultaneously, optionally wherein the polynucleotide of interest, the gene that confers resistance to the selection agent and / or the at least a portion of the editing system are in the same construct or in two or more separate constructs.

21. The method of any one of the claims 1-18, wherein introducing the polynucleotide of interest, the gene that confers resistance to the selection agent and / or the at least a portion of the editing system into the one or more meristem cells of the wounded tissue comprises introducing the polynucleotide of interest, the gene that confers resistance to the selection agent and / or the at least a portion of the editing system into the one or more meristems by particle bombardment (biolistics).

22. The method of any one of the preceding claims, wherein the modified Rubus spp. plant or plant part is an edited plant, optionally wherein the edited plant is produced in about 2 to about 6 months after the introducing step.

23. A method of producing a modified Rubus species (Rubus spp.) plant or plant part, the method comprising: wounding a Rubus spp. tissue comprising meristem cells to provide a wounded tissue comprising the meristem cells; introducing an editing system into one or more meristem cells of the wounded tissue to provide a transformed tissue, wherein the editing system is targeted (a) to a first region of a selection marker gene that is endogenous to the Rubus spp., and (b) to a second region of a gene of interest that is endogenous to the Rubus spp.; culturing the transformed tissue in the presence of a selection agent for which resistance to the selection agent is encoded by the selection marker gene to provide a cultured transformed tissue, wherein the selection agent is in an amount at least 1.5 times that needed to kill 90% of Rubus spp. cells; and regenerating a modified Rubus spp. plant or plant part from the cultured transformed tissue.

24. The method of claim 23, further comprising introducing, via the editing system, a first mutation into the selection marker gene to provide an edited selection marker gene that confers resistance to the selection agent and / or a second mutation into the gene of interest to provide an edited tissue.

25. The method of claim 23 or claim 24, wherein the Rubus spp. tissue is a Rubus allegheniensis, Rubus occidenlalis. Rubus odoralus. or Rubus fruticosus.

26. The method of any one of claims 23-25, wherein the Rubus spp. tissue is shoot tissue, optionally wherein the Rubus spp. tissue comprises meristem tissue and / or a node.

27. The method of any one of claims 23-26, wherein the selection agent is kanamycin and the kanamycin is present in an amount of at least about 75 mg / L, optionally wherein the kanamycin is present in an amount of at about 100 mg / L.

28. The method of any one of claims 23-26, wherein the selection agent is spectinomycin and the spectinomycin is present in an amount of at least about 150 mg / L.

29. The method of any one of claims 23-28, wherein prior to wounding the Rubus spp. tissue, the method comprises the step of culturing a Rubus spp. shoot material comprising a node in the presence of at least one plant growth regulator to produce the Rubus spp. tissue comprising meristem cells (e.g., shoot meristem cells).

30. The method of claim 29, wherein the at least one plant growth regulator comprises a cytokinin, a cytokinin-like compound, an auxin, an auxin precursor, and / or an antibiotic, optionally wherein the least one plant growth regulator comprises thidiazuron (TDZ) and hygromycin.

31. The method of claim 30, wherein the cytokinin or cytokinin-like compound is selected from thidiazuron (TDZ), 6-benzylaminopurine (BAP), kinetin, forchlorfenuron (4- CPPU), 6-(y,y-dimethylallylamino) purine (2iP), zeatin, zeatin-riboside, dihydro-zeatin, adenine, 6-isopentenyladenine (IP A), and / or TIBA (2,3,5-triiodobenzoic acid).

32. The method of any one of claims 29-31, wherein culturing the Rubus spp. shoot material comprising the node in the presence of the at least one growth regulator to produce the Rubus spp. tissue comprising meristem cells (e.g., shoot meristem cells) comprises culturing the Rubus spp. shoot material in the presence of thidiazuron (TDZ) in an amount of about 0.1 mg / L to about 5 mg / L and in the presence of hygromycin in an amount of about 1 mg / L to about 10 mg / L.

33. The method of any one of claims 23-32, wherein the Rubus spp. tissue comprising meristem cells (e.g., shoot meristem cells) comprises a proliferating meristem tissue and / or a proliferating shoot.

34. The method of any one of claims 23-33, wherein the transformed tissue and / or the cultured transformed tissue comprises a transgenic cell, optionally wherein the transformed tissue is a transformed shoot, optionally wherein the transgenic cell is an edited cell.

35. The method of any one of claims 23-34, wherein the transformed tissue and / or the cultured transformed tissue comprises a wild-type cell and a transgenic cell optionally wherein the transformed tissue is a transformed shoot.

36. The method of any one of claims 23-35, wherein the transformed tissue and / or the cultured transformed tissue comprises at least two different edited cells optionally wherein the transformed tissue is a transformed shoot.

37. The method of any one of claims 23-36, wherein the regenerating step comprises culturing the cultured transformed tissue on a proliferation medium to produce a shoot and subsequently culturing the shoots on root induction medium to provide the modified Rubus spp. plant or plant part.

38. The method of claim 37, wherein the modified Rubus spp. plant or plant part is mosaic.

39. The method of claim 37, wherein the modified Rubus spp. plant or plant part is nonmosaic.

40. The method of any one of claims 23-39, wherein introducing the editing system into the one or more meristem cells of the wounded tissue comprises contacting the wounded tissue with an Agrobacterium that comprises the editing system.

41. The method of claim 40, wherein the wounding and contacting steps are carried out simultaneously, optionally wherein the editing system is in a single construct or in two or more separate constructs.

42. The method of any one of the claims 23-39, wherein introducing the editing system into the one or more meristem cells of the wounded tissue comprises introducing the editing system into the one or more meristems by particle bombardment (biolistics).

43. The method of any one of claims 23-42, wherein the modified Rubus spp. plant or plant part is an edited plant, optionally wherein the edited plant is produced in about 2 to about 6 months after the introducing step.

44. The method of any one of claims 1-43, wherein the selection agent is not hygromycin.

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