Methods and compositions for reducing thorns or prickles in plants.

Description

"METHODS AND COMPOSITIONS FOR REDUCING THORNS OR PRICKS IN PLANTS" DECLARATION REGARDING THE ELECTRONIC DEPOSIT OF A SEQUENCE LISTING

[001] A Sequence Listing in XML text format, entitled 1499-127_ST26.xml, with a size of 372,120 bytes, generated on March 23, 2024 and archived here, is incorporated by reference into the specification for its disclosures. FIELD OF THE INVENTION

[002] This invention relates to compositions and methods for modifying a functional Homebox gene related to WUSCHEL (WUS) (WOX) in a Rosaceae plant, optionally to modify the architecture of the spines and / or prickles, to reduce the number and / or size of the spines, or to eliminate spines and / or prickles. The invention also relates to Rosaceae plants with spines and / or prickles with modified architecture, reduced number and / or size of spines and / or prickles, or without spines and / or prickles produced using the methods and compositions of the invention. The invention also relates to methods of identifying and / or improving plants with spines and / or prickles with modified architecture, reduced number and / or size of spines and / or prickles, or absence of spines and / or prickles. BACKGROUND OF THE INVENTION

[003] Structures such as trichomes, spines, hairs, thorns, and prickles are found on the surface of plant parts, such as stems, leaves, and shoots of many plant species. These structures have various developmental origins, such as emerging from epidermal cells that form the outermost layers of stem tissues or forming from vascular cells that are deeper within the plant tissues. Trichomes, hairs, spines, prickles are Petition 870250103857, dated 11 / 13 / 2025, page 11 / 212 2 / 166 are considered to have the common function of preventing herbivory of plant leaves and stems, as well as immature shoots. However, in agricultural and horticultural environments, thorns cause significant problems for those who handle the plants. Thorns and spines interfere with the propagation, care, and harvesting of these crops, resulting in increased costs. Genetic improvement has been used to develop some plant varieties with reduced thorn / spine production or prickles / thorns with modified architecture, but this is usually associated with other less desirable characteristics. Thus, improved strategies are needed for the development of plants with thorns and / or prickles reduced in number, reduced size, or modified architecture. SUMMARY OF THE INVENTION

[004] One aspect of the invention provides a Rosaceae plant or part of a plant comprising an endogenous mutation of the WUSCHEL-related Homebox gene (WUS) (WOX), wherein the endogenous mutant WOX gene is provided by and / or includes at least one mutation in at least one (e.g., one or more) functional endogenous WOX gene encoding a WOX transcription factor, optionally wherein at least one mutation may be an unnatural mutation.

[005] A second aspect of the invention provides a Rosaceae plant cell comprising an editing system, the editing system comprising: (a) a CRISPR-Cas effector protein; and (b) a guide nucleic acid comprising a spacer sequence with complementarity to a functional target gene encoding a WUSCHEL-related Homebox (WUS) (WOX) transcription factor in the Rosaceae plant cell.

[006] A third aspect of the invention provides a plant cell Petition 870250103857, dated 11 / 13 / 2025, page 12 / 2123 / 166 such of Rosaceae comprising at least one mutation within an endogenous WOX gene, wherein at least one mutation is a base substitution, a base insertion or a base deletion that is introduced using an editing system comprising a nucleic acid binding domain that binds to a target site within the endogenous WOX gene, wherein the endogenous WOX gene: (a) comprises a nucleotide sequence with at least 80% sequence identity with any of the SEQ ID NOs: 69, 70, 75, 76, 128, 129 or 162-173; (b) comprises a region with at least 80% sequence identity with a nucleotide sequence of any of the SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135; (c) encodes an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 71, 77, 130, or 174-182;and / or (d) encodes a region of an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 136 or SEQ ID NO: 137, optionally where at least one mutation may be an unnatural mutation.

[007] A fourth aspect of the invention provides a method for producing / improving an edited and transgene-free Rosaceae plant, comprising: crossing a Rosaceae plant of the invention with a transgene-free plant, thereby introducing at least one mutation into the Rosaceae plant that is transgene-free; and selecting a Rosaceae parent plant comprising at least one mutation and being transgene-free, thereby producing an edited and transgene-free Rosaceae plant, optionally wherein at least one mutation may be an unnatural mutation.

[008] A fifth aspect of the invention provides a method for providing a plurality of Rosaceae plants having the phenotype of a Petition 870250103857, dated 11 / 13 / 2025, p. 13 / 212 4 / 166 reduced number of thorns and / or prickles, thorns and / or prickles that are reduced in size, thorns and / or prickles having a modified architecture and / or no thorns and / or prickles, the method comprising planting two or more Rosaceae plants of the invention in a growing area, thus providing a plurality of Rosaceae plants having a phenotype of reduced number of thorns and / or prickles, thorns and / or prickles that are reduced in size, thorns and / or prickles having a modified architecture and / or no thorns and / or prickles compared with a plurality of control Rosaceae plants devoid of at least one mutation, optionally wherein at least one mutation may be an unnatural mutation.

[009] In a sixth aspect, a method is provided for creating a mutation in an endogenous Homebox gene related to WUSCHEL (WUS) (WOX) in a Rosaceae plant, the method comprising: (a) targeting a gene editing system to a portion of the endogenous WOX gene that (i) comprises a sequence with at least 80% sequence identity with any of the SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135; and / or (ii) encodes a sequence with at least 80% identity to any of the SEQ ID NOs: 126, 127, 136 or 137, and (b) select a Rosaceae plant comprising a modification located in a region of the endogenous WOX gene with at least 80% sequence identity to any of the SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135.

[0010] In a seventh aspect, a method is provided for generating variation in a WUSCHEL-related Homebox (WUS) (WOX) transcription factor in a Rosaceae plant cell, comprising: introducing an editing system into a Rosaceae plant cell, wherein the editing system is directed to a region of a functional WUSCHEL-related Homebox (WUS) (WOX) gene; Petition 870250103857, dated 11 / 13 / 2025, page 14 / 212 5 / 166 and by contacting the functional WOX gene region with the editing system, thus introducing a mutation in the functional WOX gene and generating variation in the WOX transcription factor in the Rosaceae plant cell.

[0011] An eighth aspect provides a method for editing a specific site in the genome of a Rosaceae plant cell, the method comprising: cleaving, in a site-specific manner, a target site within an endogenous Homebox-related WUSCHEL (WUS) gene (WOX) in the Rosaceae plant cell, the endogenous WOX gene: (a) comprising a nucleotide sequence with at least 80% sequence identity with any of the SEQ ID NOs: 69, 70, 75, 76, 128, 129 or 162-173; (b) comprising a region with at least 80% sequence identity with a nucleotide sequence of any of the SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135; (c) encode an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 71, 77, 130, or 174-182;and / or (d) encode a region of an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 126, 127, 136 or 137, thereby generating an edit in the endogenous WOX gene of the Rosaceae plant cell and producing a Rosaceae plant cell comprising the edit in the endogenous WOX gene.

[0012] A ninth aspect provides a method for producing a Rosaceae plant, the method comprising (a) contacting a population of Rosaceae plant cells comprising a WUSCHEL-related Homebox gene (WUS) (WOX) with a nuclease linked to a nucleic acid binding domain (e.g., editing system) that binds to a sequence (i) having at least 80% sequence identity with a nucleotide sequence of any of the SEQ ID NOs: 69, 70, 75, 76, 128, 129, or 162 Petition 870250103857, dated 13 / 11 / 2025, p. 15 / 212 6 / 166 173; (ii) comprising a region with at least 80% identity with any of the SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135; (iii) encoding an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 71, 77, 130, or 174-182, and / or (iv) encoding a region with at least 80% sequence identity with any of the SEQ ID NOs: 126, 127, 136 or 137; (b) selecting a Rosaceae plant cell from the Rosaceae plant cell population in which an endogenous WOX gene has been mutated, thereby producing a Rosaceae plant cell comprising a mutation in the endogenous WOX gene; and (c) the growth of the selected Rosaceae plant cell in a Rosaceae plant that has a mutation in the endogenous WOX gene.

[0013] A tenth aspect provides a method for reducing the number and / or size of spines, modifying the architecture of spines and / or prickles and / or eliminating spines and / or prickles (e.g., without spines and / or prickles) in a Rosaceae plant, comprising (a) contacting a Rosaceae plant cell comprising a WUSCHEL-related endogenous Homebox gene (WUS) (WOX) with a nuclease targeting the endogenous WOX gene, wherein the nuclease is linked to a nucleic acid binding domain (e.g., editing system) that binds to a target site within the endogenous WOX gene, wherein the endogenous WOX gene: (i) comprises a nucleotide sequence with at least 80% sequence identity with any of the SEQ ID NOs: 69, 70, 75, 76, 128, 129 or 162-173; (ii) comprises a region with at least 80% sequence identity with a nucleotide sequence of any of the following SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135;(iii) encodes an amino acid sequence with at least 80% sequence identity with any of the SEQs; Petition 870250103857, dated 11 / 13 / 2025, p. 16 / 212 7 / 166 ID NOs: 71, 77, 130, or 174-182; and / or (iv) encodes a region of an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 126, 127, 136, or 137 to produce a Rosaceae plant cell comprising a mutation in the endogenous WOX gene; and (b) the growth of the Rosaceae plant cell comprising a mutation in the endogenous WOX gene into a Rosaceae plant comprising the mutation in the endogenous WOX gene, thus producing a Rosaceae plant with an endogenous WOX gene mutation and a reduced number of spines and / or prickles, spines and / or prickles with modified architecture, reduced size of spines and / or prickles, and / or elimination of spines and / or prickles in the Rosaceae plant.

[0014] An eleventh aspect provides a method for producing a Rosaceae plant or part thereof comprising at least one cell with an endogenous mutation of the WUSCHEL-related Homebox gene (WUS) (WOX), the method comprising contacting a target site within an endogenous WOX gene in the Rosaceae plant or part thereof with a nuclease comprising a cleavage domain and a nucleic acid binding domain, wherein the nucleic acid binding domain binds to a target site within the endogenous WOX gene, wherein the endogenous WOX gene (a) comprises a nucleotide sequence having at least 80% sequence identity with any of the SEQ ID NOs: 69, 70, 75, 76, 128, 129 or 162-173; (b) comprises a region with at least 80% sequence identity with a nucleotide sequence of any of the following SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135;(c) encodes an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 71, 77, 130, or 174-182; and / or (d) encodes a region of an amino acid sequence with at least 80%; Petition 870250103857, dated 11 / 13 / 2025, p. 17 / 212 8 / 166 sequence identity with any of the SEQ ID NOs: 126, 127, 136 or 137, thereby producing the Rosaceae plant or part thereof comprising at least one cell with a mutation in the endogenous WOX gene.

[0015] A twelfth aspect of the invention provides a method for producing a Rosaceae plant or part thereof comprising an endogenous mutation of the WUSCHEL-related Homebox gene (WOX) and exhibiting a reduced number of spines and / or prickles, spines and / or prickles having a modified architecture, spines and / or prickles that are reduced in size and / or no spines and / or prickles, the method comprising contacting a target site within an endogenous WOX gene in the Rosaceae plant or part thereof with a nuclease comprising a cleavage domain and a nucleic acid binding domain, wherein the nucleic acid binding domain binds to a target site within the endogenous WOX gene, wherein the endogenous WOX gene: (a) comprises a nucleotide sequence with at least 80% sequence identity with any of the SEQ ID NOs: 69, 70, 75, 76, 128, 129 or 162-173;(b) comprises a region with at least 80% sequence identity with a nucleotide sequence of any of the following SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135; (c) encodes an amino acid sequence with at least 80% sequence identity with any of the following SEQ ID NOs: 71, 77, 130, or 174-182; and / or (d) encodes a region of an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 126, 127, 136 or 137, thereby producing the Rosaceae plant or part thereof comprising an endogenous WOX gene plant that has a mutation and exhibits a reduced number of spines and / or prickles, spines and / or prickles that have a modified architecture, spines and / or prickles. Petition 870250103857, of 13 / 11 / 2025, p. 18 / 212; 9 / 166 leos that are reduced in size and / or without spines and / or prickles.

[0016] A thirteenth aspect provides a guide nucleic acid that binds to a target site within a WUSCHEL-related Homebox (WUS) gene (WOX), wherein the target site is in a region of the WOX gene with at least 80% sequence identity with any of the SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135.

[0017] In a fourteenth aspect, a system is provided comprising a guide nucleic acid of the invention and a CRISPR-Cas effector protein that associates with the guide nucleic acid.

[0018] A fifteenth aspect provides a gene-editing system comprising a CRISPR-Cas effector protein in association with a guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that binds to a functional endogenous WUSCHEL-related Homebox gene (WOX).

[0019] In a sixteenth aspect, a complex comprising a guide nucleic acid and a CRISPR-Cas effector protein comprising a cleavage domain is provided, wherein the guide nucleic acid binds to a target site within the WUSCHEL-related endogenous Homebox (WUS) gene (WOX), wherein the endogenous WOX gene: (a) comprises a nucleotide sequence with at least 80% sequence identity with any of the SEQ ID NOs: 69, 70, 75, 76, 128, 129 or 162-173; (b) comprises a region with at least 80% sequence identity with a nucleotide sequence of any of the SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135; (c) encodes an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 71, 77, 130, or 174-182; and / or (d) encodes a region of an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: Petition 870250103857, dated 11 / 13 / 2025, p. 19 / 212 10 / 166 126, 127, 136, or 137, and the cleavage domain cleaves a target strand in the WOX gene.

[0020] In a seventeenth aspect, an expression cassette is provided, the expression cassette comprising (a) a polynucleotide encoding the CRISPR-Cas effector protein comprising a cleavage domain and (b) a guide nucleic acid that binds to a target site within an endogenous WUSCHEL-related Homebox gene (WUS) (WOX), wherein the guide nucleic acid comprises a spacer sequence that is complementary to and binds to (i) a portion of a core acid having at least 80% sequence identity with any of the SEQ ID NOs: 69, 70, 75, 76, 128, 129 or 162-173; (ii) a portion of a nucleic acid with at least 80% sequence identity with any of the SEQ ID NOs: 7882, 83-86, 87-90, 91-109, 110-125, or 131-135; (iii) a portion of a nucleic acid encoding an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 71, 77, 130, or 174-182;and / or (iv) a portion of a nucleic acid that encodes an amino acid sequence with at least 80% identity to any of the SEQ ID NOs: 126, 127, 136 or 137.;

[0021] In another aspect, Rosaceae plants are provided comprising in their genome one or more mutations in the Homebox genes related to WUSCHEL (WUS)s (WOX) produced by the methods of the invention, optionally wherein at least one mutation may be an unnatural mutation, optionally wherein the mutant WOX gene comprises a nucleotide sequence with at least 90% sequence identity with any of the mutant WOX genes of SEQ ID NOS: 143, 144, 145, 147, 149, 151, 153 and / or 155 and / or encodes a WOX transcription factor with at least 90% sequence identity with any of the factors of Petition 870250103857, dated 11 / 13 / 2025, p. 20 / 212 11 / 166 mutated WOX transcripts of SEQ IDs: 146, 148, 150, 152, 154 and / or 156.

[0022] In a further aspect, a genetic marker for thorns and / or prickles with a modified architecture, a reduced number and / or size of thorns and / or prickles, or no thorns and / or prickles on a blackberry plant or part thereof is provided, the genetic marker comprising (a) the nucleic acid sequence of GAACTCTG located between nucleotide positions 2897 and 2898 with reference to the nucleotide position numbering of SEQ ID NO: 69 and / or (b) the nucleic acid sequence of SEQ ID NO: 143 on a blackberry plant or part thereof is provided, the genetic marker comprising (a) the nucleic acid sequence of GAACTCTG located between nucleotide positions 2897 and 2898 with reference to the nucleotide position numbering of SEQ ID NO: 69 and / or (b) the nucleic acid sequence of SEQ ID NO: 143.

[0023] In addition, a method is provided for identifying a blackberry plant or part thereof with a thorn and / or prickle phenotype with a modified architecture, a reduced number and / or size of thorns and / or prickles, or no thorns and / or prickles, the method comprising identifying in the genome of a blackberry plant cell or part thereof (a) an insertion in an endogenous Homebox gene related to WUSCHEL (WUS) (WOX), the insertion comprising the nucleic acid sequence GAACTCTG located between nucleotide positions 2897 and 2898 with reference to the nucleotide position numbering of SEQ ID NO: 69 and / or (b) the nucleic acid sequence of SEQ ID NO: 143.

[0024] A method is further provided for producing a blackberry plant with thorns and / or prickles with a modified architecture, a reduced number and / or size of thorns and / or prickles, or without thorns and / or prickles comprising crossing a plant of Petition 870250103857, dated 11 / 13 / 2025, page 21 / 212 12 / 166 blackberry comprising in its genome (a) an insertion in an endogenous Homebox gene related to WUSCHEL (WUS) (WOX), the insertion comprising the nucleic acid sequence GAACTCTG located between nucleotide positions 2897 and 2898 with reference to the nucleotide position numbering of SEQ ID NO: 69 and / or (b) the nucleic acid sequence of SEQ ID NO: 143 with itself or with another blackberry plant to produce parent plants; and selecting one or more parent plants comprising the insertion to provide selected progeny, thereby producing blackberry plants with thorns and / or prickles with a modified architecture, a reduced number and / or size of thorns and / or prickles, or no thorns and / or prickles.

[0025] Polypeptides, polynucleotides, nucleic acid constructs, expression cassettes and vectors are further provided for the production of a Rosaceae plant of this invention.

[0026] These and other aspects of the invention are presented in more detail in the invention description below. BRIEF DESCRIPTION OF THE SEQUENCES

[0027] SEQ ID NOS:1-17 are useful exemplary Cas12a amino acid sequences with this invention.

[0028] SEQ ID Nos:18-20 are examples of Cas12a nucleotide sequences useful for this invention.

[0029] SEQ ID NO:21-22 are examples of exemplary regulatory sequences that encode a promoter and an intron.

[0030] SEQ ID Nos:23-29 are examples of cytosine deaminase sequences useful for this invention.

[0031] SEQ ID NOS:30-40 are exemplary adenine deaminase amino acid sequences useful with this invention.

[0032] SEQ ID NO:41 is a sequence of the uracil-DNA glycosylase (UGI) inhibitor useful with this invention. Petition 870250103857, dated 11 / 13 / 2025, page 22 / 212 13 / 166

[0033] SEQ ID Nos:42-44 provide examples of peptide and affinity polypeptide markers useful with this invention.

[0034] SEQ ID Nos:45-55 provide examples of RNA recruitment motifs and corresponding affinity polypeptides useful with this invention.

[0035] SEQ ID Nos:56-57 are useful exemplary Cas9 polypeptide sequences with this invention.

[0036] SEQ ID Nos: 58-68 are useful exemplary Cas9 polynucleotide sequences with this invention.

[0037] SEQ ID NO: 69 and SEQ ID NO: 72 are examples of WOX1 genomic sequences from mulberry.

[0038] SEQ ID NO:70 and SEQ ID NO: 73 are examples of WOX1 genomic sequences that encode blackberry sequences.

[0039] SEQ ID NO:71 and SEQ ID NO: 74 are examples of blackberry WOX1 transcription factor sequences encoded by SEQ ID NO: 69 and SEQ ID NO:70, respectively.

[0040] SEQ ID NO: 75 is an example of the WOX23 genomic sequence of black raspberry.

[0041] SEQ ID NO: 76 is an example of a WOX23 encoding sequence for the black raspberry.

[0042] SEQ ID NO:77 is an example of a WOX23 transcription factor sequence from black raspberry, encoded by SEQ ID NO: 75 and SEQ ID NO: 76.

[0043] SEQ ID Nos: 78-90 are example portions or regions of a WOX1 genomic sequence from blackberry (Exon 1).

[0044] SEQ ID Nos: 91-109 are example portions or regions of a WOX1 genomic sequence from blackberry (Exon 2).

[0045] SEQ ID Nos: 104, 105, 109, and 110-125 are example portions or regions of a WOX23 genomic sequence from black raspberry (Exon 2). Petition 870250103857, dated 11 / 13 / 2025, page 23 / 212 14 / 166

[0046] SEQ ID NO: 126 is an example of a portion or region of a WOX1 transcription factor from blackberry.

[0047] SEQ ID NO: 127 is an example of a portion or region of a blackberry WOX1 transcription factor and a black raspberry WOX23 transcription factor.

[0048] SEQ ID NO: 128 is an example of a WOX1 genomic sequence from red raspberry.

[0049] SEQ ID NO: 129 is an example of a WOX1 sequence encoding raspberry red.

[0050] SEQ ID NO: 130 is an example of a polypeptide sequence WOX1 red raspberry, coded by SEQ ID NO: 128 and SEQ ID NO: 129.

[0051] SEQ ID NOs: 95, 96, 97, 104, 105, 111, 112, 113, 118, 119, 120, 124, 125, and 131-135 are example portions or regions of a WOX1 genomic sequence from the red raspberry (Exon 2).

[0052] SEQ ID NO: 136 and SEQ ID NO: 136 are examples of portions or regions of a red raspberry WOX1 transcription factor.

[0053] SEQ ID NO: 138-142 are example spacer sequences for nucleic acid guides useful with this invention.

[0054] SEQ ID NO: 143 is a natural mutation of the WOX1 gene.

[0055] SEQ ID Nos: 144, 145, 147, 149, 151, 153 and 155 are examples of mutated WOX1 genomic sequences edited as described here.

[0056] SEQ ID NOs: 146, 148, 150, 152, 154 and 156 are examples of mutated WOX1 polypeptides, which are encoded by SEQ ID NOs: 145, 147, 149, 151, 153 and 155, respectively.

[0057] SEQ ID NO: 157-159 are consecutive nucleotides deleted from SEQ ID NO: 69.

[0058] SEQ ID NO: 160 is the amino acid sequence of the motif Petition 870250103857, dated 11 / 13 / 2025, p. 24 / 212 15 / 166 WUS-box from the WOX family.

[0059] SEQ ID NO: 161 is the consensus amino acid sequence of the WOX family WUS-box motif.

[0060] SEQ ID NO: 162-173 are examples of WOX1 nucleotide sequences from Rosa chinensis.

[0061] SEQ ID NO: 174-182 are examples of WOX1 amino acid sequences from Rosa chinensis. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] FIG. 1 shows a schematic of the WOX1 protein domain structure. The homeodomain (HD) is the most prominent and defining feature of the WOX family. The WOX family's WUS-box (oval) motif is strictly defined as TL-[DEQP]-LFP[GITVL]-[GSKNTCV] (SEQ ID NO: 160), consensus TLELFPLH (SEQ ID NO: 161). DETAILED DESCRIPTION

[0063] The present invention will be described below with reference to the accompanying drawings and examples, which show embodiments of the invention. This description is not intended to be a detailed catalog of all the different ways in which the invention can be implemented, or of all the features that can be added to the immediate invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a specific embodiment may be excluded from that embodiment. Thus, the invention contemplates that in some embodiments of the invention, any feature or combination of features set forth in this document may be excluded or omitted. Furthermore, numerous variations and additions to the various embodiments suggested in this document will be apparent to those skilled in the art in light of the present disclosure, which do not depart from the present invention. Therefore, the Petition 870250103857, dated 11 / 13 / 2025, p. 25 / 212 16 / 166 The following descriptions are intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.

[0064] Unless otherwise defined, all technical and scientific terms used in this document have the same meaning commonly understood by one skilled in the art to which this invention pertains. The terminology used in describing the invention in this document is for the purpose of describing only particular embodiments and is not intended to be limiting of the invention.

[0065] All publications, patent applications, patents and other references cited in this document are incorporated by reference in their entirety for the purposes relevant to the sentence and / or paragraph in which the reference is made.

[0066] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described in this document may be used in any combination. Furthermore, the present invention also contemplates that, in some embodiments of the invention, any feature or combination of features set forth in this document may 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 one of A, B, or C, or a combination thereof, may be omitted and rejected individually or in any combination.

[0067] As used in the invention description and appended claims, the singular forms a, an and the / a should also include the plural forms, unless the context clearly indicates otherwise.

[0068] 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 absence of combinations when in Petition 870250103857, dated 11 / 13 / 2025, page 26 / 212 17 / 166 interpreted in the alternative (or).

[0069] The term approximately, as used herein when referring to a measurable value, such as a quantity or concentration and the like, is intended 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, approximately X, where X is the measurable value, shall include X as well as variations of ±10%, ±5%, ±1%, ±0.5% or even ±0.1% of X. An interval given herein for a measurable value may include any other interval and / or individual value contained therein.

[0070] 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.

[0071] The citation of value ranges contained in this document is intended only as a shorthand method of individually referencing each separate value within the range, unless otherwise indicated in this document, and each separate value is incorporated into the specification as if it were individually recited in this document. For example, if the range 10 to 15 is disclosed, then 11, 12, 13, and 14 will also be disclosed.

[0072] The terms comprehend, comprehends and comprehending, as used in this document, specify the presence of the declared resources, integers, steps, operations, elements and / or components, but do not preclude the presence or addition of one or more other resources, integers, steps, operations, elements, components and / or groups thereof.

[0073] As used herein, the transition phrase consisting essentially of means that the scope of a claim should be interpreted to cover the materials or steps specified. Petition 870250103857, dated 11 / 13 / 2025, page 27 / 212 18 / 166 in the claim and those that do not materially affect the basic and novel features of the claimed invention. Therefore, the term consisting essentially of, when used in a claim of this invention, is not intended to be interpreted as equivalent to comprising.

[0074] As used herein, the terms increase, increasing, augmented, improve, improved, enhancing and improvement (and grammatical variations thereof) describe an increase of at least about 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more compared to a control.

[0075] As used in this document, the terms reduce, reduced, reducing, reduction and decrease (and grammatical variations thereof) describe, for example, a decrease of at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 9%, or 100% compared to a control. In specific embodiments, the reduction may result in none or essentially none (i.e., an insignificant amount, for example, less than about 10% or even 5%) of detectable activity or quantity. As an example, a Rosaceae plant containing a mutation in a Homebox gene related to WUSCHEL (WUS) (WOX) as described herein may exhibit a reduction in thorns and / or prickles of at least 50% when compared to a control Rosaceae plant lacking at least one mutation (e.g., a reduction in thorns and / or prickles of at least about 50% to about 100%, e.g., 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 or 100%, or any range or value den- Petition 870250103857, dated 11 / 13 / 2025, pp. 28 / 212 19 / 166 tro deles).

[0076] A control plant of Rosaceae (e.g., a Rubus species, Rosa species, Hulthemia species, Hesperrhodos spp., a Platyrhodon spp.) is normally the same plant as the edited plant, but the control plant has not been similarly edited and therefore does not possess the mutation. A control plant may be an isogenic plant and / or a wild-type plant. Thus, a control plant may be the same breeding line, variety, or cultivar as the plant in question in which a mutation, as described in this document, is introgressed, but the control breeding line, variety, or cultivar is free of the mutation. In some embodiments, a comparison between a Rosaceae plant of the invention and a control Rosaceae plant is made under the same growing conditions, for example, the same environmental conditions (soil, hydration, light, heat, nutrients, and the like).

[0077] As used herein, the terms “express”, “expresses”, “expressed” or “expression” and the like, with respect to a nucleic acid molecule and / or a nucleotide sequence (e.g., RNA or DNA), indicate that the nucleic acid molecule and / or a nucleotide sequence is transcribed and, optionally, translated. Thus, a nucleic acid molecule and / or a nucleotide sequence may express a polypeptide of interest or, for example, a functional untranslated RNA.

[0078] A heterologous or recombinant nucleotide sequence is a nucleotide sequence not naturally associated with a host cell into which it is introduced, including multiple unnatural copies of a naturally occurring nucleotide sequence. A heterologous nucleotide / polypeptide may originate from a foreign species or, if from the same species, may be substantially modified from its native form in composition and / or genomic locus. Petition 870250103857, dated 11 / 13 / 2025, pages 29 / 212 20 / 166 by deliberate human intervention.

[0079] A native or wild-type nucleic acid, nucleotide sequence, polypeptide, or amino acid sequence refers to a naturally occurring or endogenous nucleic acid, nucleotide sequence, polypeptide, or amino acid sequence. In some contexts, a wild-type nucleic acid is a nucleic acid that is not edited as described in this document and may differ from an endogenous gene that may be edited as described in this document (e.g., a mutant endogenous gene). In some contexts, a wild-type (e.g., unedited) nucleic acid may be heterologous to the organism in which the wild-type nucleic acid is found (e.g., a transgenic organism). As an example, an “endogenous wild-type WUSCHEL (WUS) Homeobox-related (WOX) gene” is a WOX gene that occurs naturally or is endogenous to the reference organism, for example, a Rosaceae plant (e.g., Rubus spp., Rosa spp., Huithemia spp., Hesperrhodos spp.).(a Platyrhodon spp), and may be subject to modifications as described herein, after which such modified endogenous gene is no longer wild type. In some embodiments, an endogenous WOX gene may be an endogenous WOX1 gene and / or an endogenous WOX23 gene. In some embodiments, an endogenous WOX gene may be functional or non-functional. A functional endogenous WOX gene produces a functional WOX transcription factor and / or expression of the functional factor. The endogenous WOX gene provides a plant or plant part that includes thorns and / or prickles. In some embodiments, a functional endogenous WOX gene comprises a nucleotide sequence with at least 80% (e.g., 85%, 90%, 95%, or 100%) sequence identity with one or more of the following SEQ ID NOs: 69, 70, 75, 76, 78-82, 83-86, 87-90, 91-109, 110-125, 128, 129, 131-135, and 162-173 and / or encodes a se. Petition 870250103857, dated 11 / 13 / 2025, page 30 / 212 21 / 166 amino acid sequence with at least 80% (e.g., 85%, 90%, 95%, or 100%) sequence identity with one or more of the following SEQ ID NOs: 71, 77, 126, 127, 130, 136, 137, and 174182. In some embodiments, a functional WOX transcription factor may be a full-length protein (e.g., when compared to a reference WOX transcription factor that is known to be functional) and / or complexes (e.g., dimerizes) with another protein (which may be the same or different) and / or nucleic acid. In some embodiments, a functional WOX transcription factor includes all of its functional domains, such as its DNA-binding domain, transactivation domain, and homeodomain, may dimerize with itself, bind to proteins other than itself, and / or may bind to DNA. In some forms, a functional WOX transcription factor may include a homeodomain and a motif, as shown in FIG.1 and / or have a structure, activity and / or function as described in van der Graaff, et al., Genome Biology 2009, 10:248. A non-functional WOX gene (e.g., a non-functional endogenous WOX gene) produces a non-functional WOX transcription factor (e.g., a truncated protein) or no WOX transcription factor.

[0080] As used herein, the term heterozygous refers to a genetic state in which different alleles reside at corresponding loci on homologous chromosomes.

[0081] As used herein, the term homozygous refers to a genetic state in which identical alleles reside at corresponding loci on homologous chromosomes.

[0082] As used herein, the term allele refers to one of two or more different nucleotides or nucleotide sequences that occur at a specific locus.

[0083] The term “mutation” refers to point mutations (by Petition 870250103857, dated 11 / 13 / 2025, page 31 / 212 22 / 166 example, missense, or nonsense, or insertions or deletions of single base pairs resulting in frameshifts), insertions, deletions, inversions, and / or truncations. When the mutation is a substitution of one residue within an amino acid sequence for another residue, or a deletion or insertion of one or more residues within a sequence, mutations are typically described by identifying the original residue followed by the position of the residue within the sequence and the identity of the newly substituted residue. A truncation may include a truncation at the C-terminal end of a polypeptide or at the N-terminal end of a polypeptide. The truncation of a polypeptide may be the result of a deletion of the corresponding 5' or 3' end of the gene encoding the polypeptide.A frameshift mutation can occur when deletions or insertions of one or more base pairs are introduced into a gene, optionally resulting in an out-of-frame or in-frame mutation. Frameshift mutations in a gene can result in the production of a polypeptide that is longer, shorter, or the same length as the wild-type polypeptide, depending on when the first stop codon occurs after the mutant region of the gene. As an example, an out-of-frame mutation that produces a premature stop codon may produce a polypeptide that is shorter than the wild-type polypeptide or, in some embodiments, the polypeptide may be absent / undetectable. A DNA inversion is the result of the rotation of a genetic fragment within a region of a chromosome.

[0084] A null allele is a non-functional allele caused by a genetic mutation that results in the complete absence of production of the corresponding protein or produces a protein that is not functional.

[0085] A knockout mutation is a mutation in a gene that produces a non-functional protein, which may be detectable, or Petition 870250103857, dated 11 / 13 / 2025, page 32 / 212 23 / 166 no protein production.

[0086] A knock-down mutation is a mutation in a gene that provides reduced protein production and / or reduced protein activity (e.g., reduced protein function) compared to the amount and / or activity of the protein produced by the gene in the absence of the mutation.

[0087] A “recessive mutation” is a mutation in a gene that produces a phenotype when homozygous, but the phenotype is not observable when the locus is heterozygous.

[0088] A “dominant mutation” is a mutation in a gene that produces a mutant phenotype in the presence of a non-mutant copy of the gene. A dominant mutation can be a loss-of-function or gain-of-function mutation, a hypomorphic mutation, a hypermorphic mutation, or a weak loss-of-function or weak gain-of-function mutation.

[0089] A dominant negative mutation is a mutation that produces an altered gene product (for example, having an aberrant function relative to the wild type), whose gene product negatively affects the function of the wild-type allele or gene product. For example, a dominant negative mutation may block a function of the wild-type gene product. A dominant negative mutation may also be called an antimorphic mutation.

[0090] A semidominant mutation refers to a mutation in which the penetrance of the phenotype in a heterozygous organism is less than that observed in a homozygous organism.

[0091] A weak loss-of-function mutation is a mutation that results in a gene product with partial or reduced function (partially inactivated) compared to the wild-type gene product.

[0092] A hypomorphic mutation is a mutation that results in Petition 870250103857, dated 11 / 13 / 2025, page 33 / 212 24 / 166 a partial loss of gene function, which can occur through reduced expression (e.g., reduced protein and / or reduced RNA) or reduced functional performance (e.g., reduced activity), but not a complete loss of function / activity. A “hypomorphic” allele is a semi-functional allele caused by a genetic mutation that results in the production of the corresponding protein functioning between 1% and 99% of normal efficiency.

[0093] A hypermorphic mutation is a mutation that results in increased gene product expression and / or increased gene product activity.

[0094] As used herein, an “unnatural mutation” refers to a mutation that is generated by human intervention and differs from mutations found in the same gene that occurred in nature (i.e., occurred naturally and not as a result of a modification made by a human).

[0095] A locus is a position on a chromosome where a gene, marker, or allele is located. In some embodiments, a locus may span one or more nucleotides.

[0096] As used herein, the terms desired allele, target allele, and / or allele of interest are used interchangeably to refer to an allele associated with a desired trait. In some embodiments, a desired allele may be associated with an increase or a decrease (relative to a control) of a given trait, depending on the nature of the desired phenotype.

[0097] A marker is associated with a trait when that trait is linked to it and when the presence of the marker is an indicator of whether and / or to what extent the desired trait or trait form will occur in a plant / germplasm comprising the marker. Similarly, a marker is associated with an allele or chromosome range when it is linked to Petition 870250103857, dated 11 / 13 / 2025, page 34 / 212 25 / 166 it and when the presence of the marker is an indicator of whether the allele or chromosome gap is present in a plant / germplasm that comprises the marker.

[0098] As used in this document, the terms backcross and backcrossing refer to the process by which a progeny plant is backcrossed with one of its parents one or more times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, etc.). In a backcrossing scheme, the donor parent refers to the parental plant with the desired gene or locus to be introgressed. The recipient parent (used one or more times) or recurrent parent (used two or more times) refers to the parental plant into which the gene or locus is being introgressed. For example, see Ragot, M. et al. Markerassisted Backcrossing: A Practical Example, in Techniques et Utilisations des Marqueurs Moleculaires Les Colloques, Vol. 72, pp. 45-56 (1995); and Openshaw et al., Marker-assisted selection in backcross breeding, in Proceedings of the Symposium Analysis of Molecular Marker Data, pp. 41-43 (1994). The initial cross gives rise to the F1 generation.The term BC1 refers to the second use of the recurring parent, BC2 refers to the third use of the recurring parent, and so on.

[0099] As used herein, the terms cross or crossbreeding refer to the fusion of gametes through pollination to produce offspring (e.g., cells, seeds, or plants). The term encompasses both sexual crossbreeding (the pollination of one plant by another) and self-fertilization (self-pollination, for example, when the pollen and ovule are from the same plant). The term crossbreeding refers to the act of fusing gametes through pollination to produce offspring.

[00100] As used in this document, the terms introgression, introgressing, and introgressed refer to the natural and artificial transmission of a desired allele or combination of desired alleles. Petition 870250103857, dated 11 / 13 / 2025, page 35 / 212 26 / 166 of a genetic locus or genetic loci from one genetic background to another. For example, a desired allele at a specific locus can be transmitted to at least one (e.g., one or more) offspring through sexual crossing between two parents of the same species, where at least one parent has the desired allele in their genome. Alternatively, for example, the transmission of an allele can occur by recombination between two donor genomes, for example, in a fused protoplast, where at least one of the donor protoplasts has the desired allele in its genome. The desired allele can be a selected allele from a marker, a QTL, a transgene, or similar. The offspring containing the desired allele can be backcrossed one or more times (e.g., 1, 2, 3, 4 or more times) with a lineage that has a desired genetic background, selecting the desired allele, with the result that the desired allele becomes fixed in the desired genetic background.For example, a marker associated with increased yield under water-free conditions could be introgressed from a donor to a recurrent parent that does not possess the marker and does not exhibit increased yield under water-free conditions. The resulting offspring could then be backcrossed one or more times and selected until the progeny possessed the genetic marker(s) associated with increased productivity under water-free conditions in the recurrent parental context.

[00101] A genetic map is a description of the genetic linkage relationships between loci on one or more chromosomes within a given species, usually represented in diagram or table form. For each genetic map, the distances between loci are measured by the recombination frequencies between them. Recombination between loci can be detected using a variety of markers. A genetic map is a product of the mapped population, of Petition 870250103857, dated 11 / 13 / 2025, page 36 / 212 27 / 166 types of markers used and the polymorphic potential of each marker between different populations. The order and genetic distances between loci may differ from one genetic map to another.

[00102] As used herein, the term genotype refers to the genetic makeup of an individual (or group of individuals) at one or more genetic loci, in contrast to the observable and / or detectable and / or manifested characteristic (the phenotype). The genotype is defined by the alleles at one or more known loci that the individual has inherited from its parents. The term genotype can be used to refer to the genetic makeup of an individual at a single locus, at multiple loci, or, more generally, the term genotype can be used to refer to the genetic makeup of an individual for all genes in its genome. Genotypes can be characterized indirectly, for example, using markers, and / or directly characterized by nucleic acid sequencing.

[00103] As used herein, the term germplasm refers to the genetic material of or from an individual (e.g., a plant), a group of individuals (e.g., a lineage, variety, or family of plants), or a clone derived from a lineage, variety, species, or culture. Germplasm may be part of an organism or cell, or it may be separate from the organism or cell. In general, germplasm provides genetic material with a specific genetic composition that provides a basis for some or all of the heritable qualities of an organism or cell culture. As used herein, germplasm includes cells, seeds, or tissues from which new plants can be grown, as well as plant parts that can be grown into a whole plant (e.g., leaves, stems, buds, roots, pollen, cells, etc.).

[00104] As used in this document, the terms cultivar and variety refer to a group of similar plants that, by Petition 870250103857, dated 11 / 13 / 2025, page 37 / 212 28 / 166 structural or genetic and / or performance characteristics can be distinguished from other varieties within the same species.

[00105] As used in this document, the terms exotic, exotic lineage, and exotic germplasm refer to any plant, lineage, or germplasm that is not elite. In general, exotic plants / germplasms are not derived from any known elite plant or germplasm, but are selected to introduce one or more desired genetic elements into a breeding program (e.g., to introduce new alleles into a breeding program).

[00106] As used herein, the term hybrid in the context of plant breeding refers to a plant that is the offspring of genetically different parents, produced by crossing plants of different lines, races or species, including, but not limited to, crossing between two inbred lines.

[00107] As used herein, the term inbred refers to a substantially homozygous plant or variety. The term may refer to a plant or plant variety that is substantially homozygous across the entire genome or that is substantially homozygous with respect to a part of the genome that is of particular interest.

[00108] A haplotype is the genotype of an individual across a plurality of genetic loci, that is, a combination of alleles. Typically, the genetic loci that define a haplotype are physically and genetically linked, that is, on the same chromosomal segment. The term haplotype can refer to polymorphisms at a specific locus, such as a single marker locus, or polymorphisms at multiple loci along a chromosomal segment.

[00109] A Rosaceae plant in which at least one (e.g., one or more, for example, 1, 2, 3 or 4 or more) functional gene Petition 870250103857, dated 11 / 13 / 2025, p. 38 / 212 29 / 166 endogenous WOX (e.g., a functional endogenous WOX1 gene and / or a functional endogenous WOX23 gene) is modified as described herein (e.g., comprises a modification as described herein) may have a reduced number of spines and / or prickles, spines and / or prickles that have a modified architecture, spines and / or prickles that are reduced in size, and / or no spines and / or prickles when compared to a control Rosaceae plant lacking at least one mutation.

[00110] As used herein, thorn refers to a smaller version of a spine and can be described as a short, thin, pointed protrusion projecting from the outer surface or epidermis of a plant. As used herein, a spine means a woody, sharp, rigid, straight or curved projection. As used herein, a trichome refers to a hair-like structure projecting from the surface or epidermis of a plant. As used herein, a “hair” means a thin, pointed, flexible structure projecting from the surface or epidermis of a plant. The terms spine, spines, trichomes, and hairs may refer to the same type of plant structure and may vary depending on the rigidity, sharpness, and overall architecture of the plant structure. Plant hairs are commonly believed to be less rigid, less pointed, and less sharp than a spine or thorn.All of these terms can be used to describe structures that have more than one pointed protrusion and may contain up to 10 pointed structures emerging from a central point in the plant's epidermis.

[00111] The terms thorn and spine (and grammatical variations thereof) will be used interchangeably here. Thus, although in blackberry, thorns are more precisely called thorns or spines, they may be called thorns or spines here, with the absence of thorns and / or prickles being described Petition 870250103857, dated 11 / 13 / 2025, page 39 / 212 30 / 166 ta como sem espinhos, sem espinhos, sem espinhos ou sem espinhas.

[00112] As used herein, reduced number of thorns and / or prickles means a reduction in thorns and / or prickles of at least 5% when compared to a control Rosaceae plant lacking at least one mutation (e.g., a reduction in the number of thorns and / or prickles of at least about 5% to about 100%, for example, a reduction of about 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, 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 or 100%, or any range or value therein). In some modalities, a reduced number of thorns and / or prickles means a reduction in thorns and / or prickles of at least 50% when compared to a control Rosaceae plant lacking at least one mutation (for example, a reduction in the number of thorns and / or prickles of at least about 50% to about 100%, for example, a reduction of 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 or 100%, or any range or value contained therein).In some embodiments, the reduction in the number of spines and / or thorns means a reduction of spines and / or thorns of at least 75% (for example, 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 100%, or any range or value therein).

[00113] Substantially spineless or substantially thornless, as used herein, refers to having a reduction in the amount of spines and / or thorns by approximately 50%, 51%, 52%, Petition 870250103857, dated 11 / 13 / 2025, p. 40 / 212 31 / 166 53%, 54%, 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% or 99.5%.

[00114] As used herein, a reduction in the size of thorns and / or prickles means a reduction in size of at least about 10% when compared to the thorns and / or prickles of a control Rosaceae plant lacking at least one mutation (e.g., a reduction in the size of thorns and / or prickles of at least about 10% to about 100%, for example, a reduction of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 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, or 100%, or any interval or value contained therein), where a reduction of 100% means that the thorn or splinter being measured is absent. When all the thorns and splinters of a plant are reduced by 100%, then the plant can be considered thornless or splintless. When all the thorns and splinters of a specific part of the plant, such as a stem or leaf, are reduced by 100%, then the plant can be considered "thornless or splintless".

[00115] In some embodiments, the architecture of a thorn or spine may be modified. A modified architecture, as used herein, means any change in physical structure, such as reduction in size, rigidity, number of spines or sharp points on a thorn, sharpness, thickness of a spine, thickness of sharp point(s) on a spine, thickness of the region where a thorn / spine attaches to the stem. A modified architecture may include Petition 870250103857, dated 11 / 13 / 2025, page 41 / 212 32 / 166 a thorn or point that is reduced in strength (for example, a thorn or point that bends like a hair instead of being rigid compared to a thorn or point that is not modified). A thorn or point with modified architecture can cause the thorn or point to be less sharp. A thorn or point with modified architecture can cause the thorn or point to be less sharp and hard or rigid.

[00116] As used herein, a control plant means a Rosaceae plant that does not contain an edited WOX gene or genes (e.g., WOX1, WOX23) as described herein that confers an altered phenotype of reduced number of spines and / or prickles, spines and / or prickles that have a modified architecture, spines and / or prickles that are reduced in size and / or no spines and / or prickles. A control Rosaceae plant is used to identify and select an edited Rosaceae plant as described herein that has an enhanced trait or altered phenotype compared to the control Rosaceae plant. A suitable control plant may be a plant from the parental line used to generate a plant comprising a mutation in the WOX gene(s), for example, a wild-type plant devoid of an edit in an endogenous WOX gene as described herein.A suitable control plant may also be a plant containing recombinant nucleic acids that confer other characteristics, for example, a transgenic plant with increased herbicide tolerance. A suitable Rosaceae control plant may, in some cases, be an offspring of a heterozygous or hemizygous Rosaceae transgenic plant line that does not possess the mutation in the WOX gene as described here, known as a negative segregant or negative isogenic line.

[00117] Here too, the term characteristic modification Petition 870250103857, dated 11 / 13 / 2025, page 42 / 212 33 / 166 covers the alteration of a natural trait, producing a detectable difference in a trait of a Rosaceae plant, comprising a mutation in an endogenous functional WOX gene as described herein in relation to a Rosaceae plant that does not comprise the mutation, such as a wild-type plant or a negative segregant. In some cases, the trait modification can be assessed quantitatively. For example, a trait modification may imply an increase or decrease in an observed trait or phenotype compared to a control plant. It is known that there may be natural variations in a modified trait. Therefore, the observed trait modification may imply a change in the normal distribution and magnitude of the trait or phenotype characteristics in plants compared to a control plant.

[00118] This disclosure relates to a Rosaceae plant with economically relevant enhanced characteristics, more specifically a reduced number of thorns and / or prickles, thorns and / or prickles having a modified architecture including, but not limited to, reduced size, reduced rigidity or hardness, reduced sharpness and the like, and / or absence of thorns and / or prickles. More specifically, this disclosure relates to a Rosaceae plant comprising one or more mutations in functional WOX gene(s) (e.g., functional WOX1, functional WOX23) as described herein, wherein the Rosaceae plant has a reduced number of thorns and / or prickles, thorns and / or prickles having a modified architecture, thorns and / or prickles that are reduced in size and / or no thorns and / or prickles compared to a control Rosaceae plant lacking the said mutation(s).

[00119] As used herein, the terms nucleic acid, molecule Petition 870250103857, dated 11 / 13 / 2025, page 43 / 212 34 / 166 The terms nucleic acid, nucleotide sequence, and polynucleotide refer to RNA or DNA that is linear or branched, single-stranded 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 pairing, dsRNA, and ribozyme. For example, polynucleotides containing C-5 propyne analogs of uridine and cytidine have been shown to bind RNA with high affinity and be potent antisense inhibitors of gene expression. Other modifications, such as modification of the phosphodiester structure or the 2'-hydroxyl group in the ribose sugar of RNA, can also be made.

[00120] As used herein, the term nucleotide sequence refers to a heteropolymer of nucleotides or to the sequence of such nucleotides from the 5' to 3' end of a nucleic acid molecule and includes DNA or RNA molecules, including cDNA, a fragment or portion of DNA, genomic DNA, synthetic (e.g., chemically synthesized) DNA, plasmid DNA, mRNA, and antisense RNA, any of which may be single-stranded or double-stranded. The terms nucleotide sequence, nucleic acid, nucleic acid molecule, nucleic acid construct, oligonucleotide, and polynucleotide are also used interchangeably in this document to refer to a heteropolymer of nucleotides.The nucleic acid molecules and / or nucleotide sequences provided herein are presented in the 5' to 3' direction, left to right, and are represented using the standard code for representing nucleotide characters as set forth in the U.S. Sequencing Rules, 37 CFR §§1.831–1.835 and the World Intellectual Property Organization (WIPO) Standard ST.26. A 5' region, as used in this document, may signify the region of a polynucleotide. Petition 870250103857, dated 11 / 13 / 2025, page 44 / 212 35 / 166 refers to the region closest to the 5' end of the polynucleotide. Thus, for example, an element in the 5' region of a polynucleotide can be located anywhere from the first nucleotide located at the 5' end of the polynucleotide to the nucleotide located in the middle of the polynucleotide. A 3' region, as used in this document, can mean the region of a polynucleotide that is closest to the 3' end of the polynucleotide. Thus, for example, an element in the 3' region of a polynucleotide can be located anywhere from the first nucleotide located at the 3' end of the polynucleotide to the nucleotide located in the middle of the polynucleotide.

[00121] As used herein with respect to nucleic acids, the term fragment or portion refers to a nucleic acid that is reduced in relative length (e.g., reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, R. 280, 290, 300, 310, 320, 330, 340, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 or 900 or more nucleotides or any interval or value therein) to a reference nucleic acid and comprising, essentially consisting of and / or consisting of a sequence of identical or nearly identical contiguous nucleotides (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% identical) to a corresponding portion of the reference nucleic acid.Such a nucleic acid fragment may, when appropriate, be included in a larger polynucleotide of which it is a constituent. As an example, a guide nucleic acid repeat sequence of this invention may comprise a portion of a wild-type CRISPR-Cas repeat sequence (e.g., a CRISPR-Cas repeat). Petition 870250103857, dated 11 / 13 / 2025, p. 45 / 212 36 / 166 wild type; for example, a CRISPR Cas system repeat of, for example, a Cas9, Cas12a (Cpf1), Cas12b, Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12g, Cas12h, Cas12i, C2c4, C2c5, C2c8, C2c9, C2c10, Cas14a, Cas14b and / or a Cas14c and the like).

[00122] In some embodiments, a nucleic acid fragment may comprise, consist essentially of, or consist of about 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, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285 290, 295, 300, 305, 310, 320, 330, 340, 350, 360, 370, 380, 390, 395, 400, 410, 415, 420, 425, 430, 435, 440, 445, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1900, 2000, 3000, 4000 or 5000 or more consecutive nucleotides, or any interval or value contained therein,of a nucleic acid that codes for a WOX polypeptide, optionally a fragment of a WOX gene (WOX1, WOX23) may have approximately 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, 115, 120, 125, 130, 135, 140, 145, 150 consecutive nucleotides for approximately 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225 230, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 340, 345, 350, 355, 360, 365, 370, 375, Petition 870250103857, dated 11 / 13 / 2025, page 46 / 212 37 / 166 380, 385, 390, 395, 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 or 750 or more consecutive nucleotides in length, or any range or value contained therein (for example, a fragment or portion of any of the SEQ ID NOs: 69, 70, 75, 76, 128, 129 or 162-173 (for example, SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally any of the SEQ ID NOs: 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 131, 132, 133, 134, and / or 135) .

[00123] As used herein with respect to polypeptides, the term fragment or portion may refer to a polypeptide that is reduced in length relative to a reference polypeptide and that comprises, consists essentially of, and / or consists of an amino acid sequence of contiguous amino acids identical or nearly identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to a corresponding portion of the reference polypeptide. Such a polypeptide fragment may, where appropriate, be included in a larger polypeptide of which it is a constituent. In some embodiments, a polypeptide fragment may comprise, consist essentially of, or consist of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 260, 270, 280, or 290 or more consecutive amino acids of a reference polypeptide.In some embodiments, a polypeptide fragment may comprise, consist essentially of, or consist of 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. Petition 870250103857, dated 11 / 13 / 2025, page 47 / 212 38 / 166 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, or 150 or more consecutive amino acid residues, or any range or value contained therein, of a WOX transcription factor (for example, a fragment or portion of any of the polypeptides with SEQ ID NOS: 71, 77, 130, or 174-182 (for example, SEQ ID NOS: 126, 127, 136, or 137)). In some embodiments, a fragment of a WOX transcription factor (WOX1 transcription factor, WOX23 transcription factor) may comprise, consist essentially of, or consist of about 20 to about 60 consecutive amino acid residues (e.g., about 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 or 60 consecutive amino acid residues, optionally about 36 or 42 residues). of consecutive amino acids, or any range or value contained therein, of a WOX transcription factor (see, for example, SEQ ID NOs: 126, 127, 136 or 137).In some embodiments, a fragment of a WOX transcription factor or a WOX polynucleotide may be the result of a deletion generated in the functional WOX gene resulting in a truncated polypeptide, optionally where the amount of the WOX transcription factor is reduced or undetectable. A deletion may result, for example, in an out-of-frame deletion allele. A functional WOX gene may be edited at more than one location, thus providing a WOX gene comprising more than one mutation (i.e., 2, 3, 4, 5, 6 or more mutations, optionally 2 or 3 mutations).

[00124] In some embodiments, a fragment of a WOX transcription factor (WOX1 transcription factor, WOX23 transcription factor) may be a truncated WOX polypeptide resulting from a Petition 870250103857, dated 11 / 13 / 2025, page 48 / 212 39 / 166 mutation of the WOX genomic sequence encoding the WOX transcription factor as described herein (e.g., a mutation in Exon 1 or Exon 2 of the functional WOX gene). In some embodiments, a fragment of a WOX transcription factor may be from the C-terminal of the WOX transcription factor. In some embodiments, a fragment of a WOX transcription factor may comprise at least a portion of the N-terminal portion of the transcription factor in addition to the C-terminal portion of the transcription factor.

[00125] In some embodiments, such exclusion, when included in a Rosaceae plant, may result in the Rosaceae plant exhibiting a reduced number of thorns and / or prickles, thorns and / or prickles having a modified architecture, thorns and / or prickles that are reduced in size, and / or no thorns and / or prickles compared to a Rosaceae plant that does not include (lack) such exclusion. A functional WOX gene can be edited in one or more locations (and using one or more different editing tools), thus providing a WOX gene that includes one or more mutations. In some embodiments, a functional WOX gene mutated as described herein may comprise a deletion that can result in the encoded polypeptide having a deletion of one or more amino acid residues (e.g., a deletion of 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 or more consecutive amino acid residues and any range or value on them (e.g., a truncated polypeptide), optionally a deletion of about 100 to about 350 consecutive amino acid residues (e.g., about 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, Petition 870250103857, dated 11 / 13 / 2025, p. 49 / 212 40 / 166 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345 or 350 consecutive amino acid residues and any range or value contained therein).

[00126] In some embodiments, a portion or region referring to a nucleic acid means at least 2, 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, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 285, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500 or 5000 or more consecutive nucleotides of a gene (e.g., consecutive nucleotides of a WOX gene), optionally a portion or region of a WOX gene may be approximately 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, 101, 102, 103, 104, 105, 110, 111, 112, 113, 114, 115, 120, 125, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 150, 151, 152, 153, 154, 155, 160, 165, 170, 171 172, 173, 174, 175, 176, 177, 178, 179, 180, 185, 190, 191, 192, 193, 194, 195, 200, 205, 210, 215, 216, 217, 218, 219, 220, 221, 222, 223,224, 225, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 245, 250, 251, 252, 253, 254, 255, 256, 257, 258, Petition 870250103857, dated 11 / 13 / 2025, p. 50 / 212 41 / 166 259, 260, 265, 270, 271, 273, 274, 275, 280, 285, 290, 295, 300, 305, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 325, 326, 327, 328, 329, 330, 331, 33, 233, 335, 336, 337, 338, 339, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, or 450 or more consecutive nucleotides in length, or any range or value contained therein (for example, a portion or region of any of the SEQ ID NOs: 69, 70, 75, 76, 128, 129, or 162-173 (for example, SEQ ID NOs: 78-125 or 131-135)).

[00127] In some embodiments, a portion or region of a WOX polypeptide sequence may be about 5 to about 100 or more consecutive amino acid residues in length (e.g., about 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, or 100 or more consecutive amino acid residues in length, optionally about 20 to about 50 consecutive amino acid residues (e.g., a portion of any of SEQ ID NOs: 71, 77, 130, or 174-182, optionally SEQ ID NOs: 126, 127, 136, or 137).

[00128] In some embodiments, a sequence-specific nucleic acid binding domain may bind to one or more fragments or portions of nucleotide sequences (e.g., DNA, RNA) that encode, for example, a WUSCHEL (WUS) - Homeobox related (WOX) transcription factor as described herein.

[00129] As used herein with respect to nucleic acids, the term functional fragment refers to the nucleic acid that encodes a functional fragment of a polypeptide. A “functional fragment” Petition 870250103857, dated 11 / 13 / 2025, page 51 / 212 42 / 166 relative to a polypeptide is a fragment of a polypeptide that retains one or more activities of the native reference polypeptide.

[00130] The term gene, as used herein, refers to a nucleic acid molecule capable of being used to produce mRNA, antisense RNA, miRNA, antisense oligodeoxyribonucleotide (AMO), anti-microRNA, and the like. Genes may or may not be capable of being used to produce a functional protein or gene product. Genes may include coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences, and / or untranslated 5' and 3' regions). A gene may be isolated, meaning a nucleic acid that is substantially or essentially free of components normally found in association with the nucleic acid in its natural state. These components include other cellular material, recombinant production culture medium, and / or various chemicals used in the chemical synthesis of the nucleic acid.

[00131] The terms complementary or complementarity, as used in this document, refer to the natural linking of polynucleotides under permissive salt and temperature conditions by base pairing. For example, the sequence AGT (5' to 3') links to the complementary sequence TCA (3' to 5'). Complementarity between two single-stranded molecules can be partial, in which only some nucleotides link, or it can be complete, when there is total complementarity between the single-stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.

[00132] Complement, as used herein, may mean 100% complementarity with the comparator nucleotide sequence or may mean less than 100% complementarity (e.g. Petition 870250103857, dated 11 / 13 / 2025, p. 52 / 212 43 / 166 plo, approximately 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% and similar, complementarity) with the comparator nucleotide sequence.

[00133] Different nucleic acids or proteins with homology are referred to here as homologs. The term homolog includes homologous sequences from the same and other species and orthologous sequences from the same and other species. “Homology” refers to the level of similarity between two or more nucleic acid and / or amino acid sequences in terms of percentage of positional identity (i.e., sequence similarity or identity). Homology also refers to the concept of similar functional properties between different nucleic acids or proteins. Thus, the compositions and methods of the invention further comprise homologs to the nucleotide sequences and polypeptide sequences of this invention. Ortholog, as used herein, refers to homologous nucleotide sequences and / or amino acid sequences in different species that arose from a common ancestral gene during speciation.A homolog of a nucleotide sequence of this invention has substantial sequence identity (for example, at least 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%, 99.5% or 100%) to said nucleotide sequence of the invention.

[00134] As used herein, sequence identity refers to the extent to which two perfectly aligned polynucleotide or polypeptide sequences are invariant over a component alignment window, for example, nucleotides or amino acids. “Identity” can be easily calculated by known methods, including, without limitation, those described in: ComputaPetition 870250103857, of 11 / 13 / 2025, p. 53 / 212 44 / 166 tional Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); e Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).

[00135] As used herein, the term sequence identity percent or identity percent refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference (consult) polynucleotide molecule (or its complementary strand) compared to a test (subject) polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned. In some embodiments, sequence identity percent may refer to the percentage of identical amino acids in an amino acid sequence compared to a reference polypeptide. With respect to a WOX1 gene, a sequence may have at least 80% sequence identity with the nucleotide sequence of one or more of the following SEQ ID NOs: 69, 70, 75, 76, 128, 129 and / or 162173.In some embodiments, a WOX1 gene may have at least 85% sequence identity with the nucleotide sequence of one or more of the following SEQ ID NOs: 69, 70, 75, 76, 128, 129, and / or 162-173. In some embodiments, a WOX1 gene may have at least 90% sequence identity with the nucleotide sequence of one or more of the following SEQ ID NOs: 69, 70, 75, 76, 128, 129, and / or 162-173. In some embodiments, a WOX1 gene may have at least 95% sequence identity with the nucleotide sequence of one or more of the following SEQ ID NOs: 69, 70, 75, 76. Petition 870250103857, dated 11 / 13 / 2025, p. 54 / 212 45 / 166 128, 129 and / or 162-173, optionally wherein the WOX1 gene may have 100% sequence identity with the nucleotide sequence of one or more of the following SEQ ID NOs: 69, 70, 75, 76, 128, 129 and / or 162-173. A WOX1 polypeptide as described herein may have at least 80% sequence identity with the polypeptide sequence of one or more of the following SEQ ID NOs: 71, 77, 130 and / or 174-182. In some embodiments, a WOX1 polypeptide may have at least 85% sequence identity with the polypeptide sequence of one or more of the following SEQ ID NOs: 71, 77, 130 and / or 174-182. In some embodiments, a WOX1 polypeptide may have at least 90% sequence identity with the polypeptide sequence of one or more of the following SEQ ID NOs: 71, 77, 130, and / or 174-182.In some embodiments, a WOX1 polypeptide may have at least 95% sequence identity with the polypeptide sequence of one or more of the following SEQ ID NOs: 71, 77, 130 and / or 174-182, optionally where the WOX1 polypeptide may have 100% sequence identity with the polypeptide sequence of one or more of the following SEQ ID NOs: 71, 77, 130 and / or 174-182. With respect to regions or portions of a WOX1 gene, the region or portion may have at least 80% sequence identity with the nucleotide sequence of any of the following SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 and / or 131-135, optionally at least 80% sequence identity with any of the following SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120. 122, 124, 125 and / or 134. In some embodiments, a region or portion of a WOX1 gene may have at least 85% sequence identity with the nucleotide sequence of any of the SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 and / or 131-135, optionally at least 85% sequence identity with any of the SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, Petition 870250103857, dated 11 / 13 / 2025, p. 55 / 212 46 / 166 107-109, 113-116, 118-120, 122, 124, 125 and / or 134. In some embodiments, a region or portion of a WOX1 gene may have at least 90% sequence identity with the nucleotide sequence of any of the SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 and / or 131-135, optionally at least 90% sequence identity with any of the SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 and / or 134. In some embodiments, a region or portion of a WOX1 gene may have at least 95% sequence identity with the nucleotide sequence of any of the following SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 and / or 131-135, optionally at least 95% sequence identity with any of the following SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 and / or 134.In some embodiments, a region or portion of a WOX1 gene may have 100% sequence identity with the nucleotide sequence of any of the following SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125, and / or 131-135, optionally 100% sequence identity with any of the following SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125, and / or 134. In some embodiments, a mutant WOX1 gene may have at least 90% sequence identity with a mutant WOX1 gene that possesses a sequence of Nucleotides from any of the SEQ ID Nos: 143, 144, 145, 147, 149, 151, 153, and / or 155. In some embodiments, a WOX1 mutant gene may have at least 95% sequence identity with a WOX1 mutant gene having a nucleotide sequence from any of the SEQ ID Nos: 143, 144, 145, 147, 149, 151, 153, and / or 155.In some embodiments, a WOX1 mutant gene may have 100% sequence identity with a WOX1 mutant gene having a nucleotide sequence of any of the following SEQ IDs: Petition 870250103857, dated 11 / 13 / 2025, p. 56 / 212 47 / 166 143, 144, 145, 147, 149, 151, 153 and / or 155. In some embodiments, a mutated WOX1 polypeptide may have at least 90% sequence identity with a mutated WOX1 polypeptide having an amino acid sequence of any of the following: SEQ ID NOs: 146, 148, 150, 152, 154 and / or 156. In some embodiments, a mutated WOX1 polypeptide may have at least 95% sequence identity with a mutated WOX1 polypeptide having an amino acid sequence of any of the following: SEQ ID NOs: 146, 148, 150, 152, 154 and / or 156. In some embodiments, a mutated WOX1 polypeptide may have 100% sequence identity with a mutant WOX1 polypeptide with an amino acid sequence of any of the following. one of the following SEQ ID numbers: 146, 148, 150, 152, 154 and / or 156.

[00136] As used herein, the phrase substantially identical or substantial identity in the context of two nucleic acid molecules, nucleotide sequences, or polypeptide sequences refers to two or more sequences or subsequences that have at least 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%, 99.5%, or 100% identity of nucleotide or amino acid residues when compared and aligned for correspondence. maximum, as measured using one of the following sequence comparison algorithms or by visual inspection.In some embodiments of the invention, substantial identity exists over a region of consecutive nucleotides of a nucleotide sequence of the invention that is from about 10 nucleotides to about 20 nucleotides, from about 10 nucleotides to about 25 nucleotides, from about 10 nucleotides to about 30 nucleotides, from about 15 nucleotides to about 25 nucleotides, from about 30 nucleotides to about 40 nucleotides, from about 50 nucleotides to about 60 nucleotides, from about 70 nucleotides to about 25. Petition 870250103857, dated 11 / 13 / 2025, page 57 / 212 48 / 166 ca of 80 nucleotides, about 90 nucleotides to about 100 nucleotides, about 100 nucleotides to about 200 nucleotides, about 100 nucleotides to about 300 nucleotides, about 100 nucleotides to about 400 nucleotides, about 100 nucleotides to about 500 nucleotides, about 100 nucleotides to about 600 nucleotides, about 100 nucleotides to about 800 nucleotides, about 100 nucleotides to about 900 nucleotides, or more in length, or any interval within it, up to the total length of the sequence. In some embodiments, the nucleotide sequences may be substantially identical in at least about 20 nucleotides (for example, about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70 or 80 nucleotides or more).

[00137] In some embodiments of the invention, substantial identity exists over a region of consecutive amino acid residues of a polypeptide of the invention that is from about 3 amino acid residues to about 20 amino acid residues, about 5 amino acid residues to about 25 amino acid residues, about 7 amino acid residues to about 30 amino acid residues, about 10 amino acid residues to about 25 amino acid residues, about 15 amino acid residues to about 30 amino acid residues, about 20 amino acid residues to about 40 amino acid residues, about 25 amino acid residues to about 40 amino acid residues, about 25 amino acid residues to about 50 amino acid residues, about 30 amino acid residues to about 50 amino acid residues, about 40 amino acid residues to about 50 amino acid residues, about 40 amino acid residues to about 70 amino acid residues,about 50 amino acid residues to about 70 amino acid residues. residues, about 60 amino acid residues about, Petition 870250103857, dated 11 / 13 / 2025, p. 58 / 212 49 / 166 of 80 amino acid residues, from about 70 amino acid residues to about 80 amino acid residues, from about 90 amino acid residues to about 100 amino acid residues, or more amino acid residues in length, and any interval therein, up to the total length of the sequence. In some embodiments, the polypeptide sequences may be substantially identical to each other in at least about 8 consecutive amino acid residues (e.g., about 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, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 130, 140, 150, 175, 200, 225, 250, 300, 350 or more amino acids in length or more consecutive amino acid residues). In some embodiments, two or more WOX transcription factors may be identical or substantially identical (e.g., at least 70% to 99.9% identical; e.g., approximately 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%, 99.5%, 99.9% identical or any range or value therein) in at least 8 consecutive amino acids to approximately 350 consecutive amino acids.In some embodiments, two or more WOX transcription factors may be identical or substantially identical in at least 8, 9, 10, 11, 12, 13, 14, or 15 consecutive amino acids to approximately 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 consecutive amino acids.

[00138] For sequence comparison, one sequence typically acts as a reference sequence against which the others are compared. Petition 870250103857, dated 11 / 13 / 2025, p. 59 / 212 50 / 166 test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, the subsequence coordinates are assigned, if necessary, and the sequence algorithm program parameters are assigned. The sequence comparison algorithm then calculates the sequence identity percentage for the test sequences with respect to the reference sequence, based on the assigned program parameters.

[00139] The ideal alignment of sequences for aligning a comparison window is well known to those skilled in the art and can be conducted by tools such as the Smith and Waterman local homology algorithm, the Needleman and Wunsch homology alignment algorithm, the Pearson and Lipman similarity search method, and optionally by computerized implementations of these algorithms, such as GAP, BESTFIT, FASTA, and TFASTA, available as part of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA). An identity fraction for aligned segments of a test sequence and a reference sequence is the number of identical components that are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, for example, the entire reference sequence or a defined smaller part of the reference sequence.The percentage identity of the sequence is represented as the identity fraction multiplied by 100. The comparison of one or more polynucleotide sequences can be with a complete polynucleotide sequence or a portion thereof, or with a longer polynucleotide sequence. For the purposes of this invention, the percentage identity can also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences. Petition 870250103857, dated 11 / 13 / 2025, page 60 / 212 51 / 166

[00140] Two nucleotide sequences can also be considered substantially complementary when the two sequences hybridize with each other under stringent conditions. In some embodiments, two nucleotide sequences considered substantially complementary hybridize with each other under highly stringent conditions.

[00141] Strict hybridization conditions and rigorous hybridization wash conditions in the context of nucleic acid hybridization experiments, such as Southern and Northern hybridizations, are sequence-dependent and differ under different environmental parameters. A comprehensive guide to nucleic acid hybridization can be found in Tijssen Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes Part I Chapter 2 Overview of principles of hybridization and the strategy of nucleic acid probe assays Elsevier, New York (1993). Generally, highly stringent hybridization and wash conditions are selected to be approximately 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.

[00142] Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes with a perfectly matching probe. Very stringent conditions are selected to match Tm for a specific probe. An example of stringent hybridization conditions for hybridizing complementary nucleotide sequences that have more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide with 1 mg heparin at 42 °C, with hybridization being performed overnight. An example of highly stringent washing conditions is 0.15 M NaCl at 72 °C for about 15 minutes. An example of stringent washing conditions is a 0.2x SSC wash at 65 °C for Petition 870250103857, dated 11 / 13 / 2025, p. 61 / 212 52 / 166 minutes (see Sambrook below for a description of the SSC buffer). Often, a high-severity wash is preceded by a low-severity wash to remove the background probe signal. An example of a medium-stringence wash for a duplex of, for example, more than 100 nucleotides, is 1x SSC at 45°C for 15 minutes. An example of a low-stringence wash for a duplex of, for example, more than 100 nucleotides, is 4-6x SSC at 40°C for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions generally involve salt concentrations less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is usually at least about 30 °C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.In general, a signal-to-noise ratio of 2x (or greater) than that observed for an unrelated probe in the specific hybridization assay indicates the detection of a specific hybridization. Nucleotide sequences that do not hybridize under stringent conditions are still substantially identical if the proteins they encode are substantially identical. This can occur, for example, when a copy of a nucleotide sequence is created using the maximum codon degeneracy allowed by the genetic code.

[00143] A recombinant polynucleotide and / or nucleic acid construct of this invention (e.g., expression cassettes and / or vectors) may have an expression-optimized codon. In some embodiments, the polynucleotides, nucleic acid constructs, expression cassettes and / or vectors of the editing systems of the invention (e.g., comprising / encoding a sequence-specific nucleic acid binding domain (e.g., a sequence-specific nucleic acid binding domain (e.g., domain of Petition 870250103857, dated 11 / 13 / 2025, page 62 / 212 53 / 166 DNA binding) of a polynucleotide-guided endonuclease, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an Argonaute protein, and / or a CRISPR-Cas endonuclease (e.g., CRISPR-Cas effector protein) (e.g., a Type I CRISPR-Cas effector protein, a Type II CRISPR-Cas effector protein, a Type III CRISPR-Cas effector protein, a Type IV CRISPR-Cas effector protein, a Type V CRISPR-Cas effector protein, or a Type VI CRISPR-Cas effector protein)), a nuclease (e.g., an endonuclease (e.g., Fok1), a polynucleotide-guided endonuclease, a CRISPR-Cas endonuclease (e.g., CRISPR-Cas effector protein), a zinc finger nuclease zinc and / or a transcription activator-like effector nuclease (TALEN), deaminase proteins / domains (e.g., adenine deaminase, cytosine deaminase), a polynucleotide encoding a reverse transcriptase protein or domain,A polynucleotide encoding a 5'-3' exonuclease polypeptide and / or affinity polypeptides, peptide markers, etc.) can be codon-optimized for expression in a plant. In some embodiments, the codon-optimized nucleic acids, polynucleotides, expression cassettes, and / or vectors of the invention have approximately 70% to approximately 99.9% (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%, 99.5%) polynucleotides, expression cassettes, and / or vectors that have not been codon-optimized.

[00144] In any of the embodiments described herein, a polynucleotide or nucleic acid construct of the invention may be operatively associated with a variety of promoters and / or other Petition 870250103857, dated 11 / 13 / 2025, p. 63 / 212 54 / 166 regulatory elements for expression in a plant and / or a plant cell. Thus, in some embodiments, a polynucleotide or nucleic acid construct of this invention may further comprise one or more promoters, introns, enhancers and / or terminators operationally linked to one or more nucleotide sequences. In some embodiments, a promoter may be operationally associated with an intron (e.g., promoter and intron Ubi1). In some embodiments, a promoter associated with an intron may be referred to as a promoter region (e.g., promoter and intron Ubi1) (see, for example, SEQ ID NO:21 and SEQ ID NO:22).

[00145] By operationally linked or operationally associated, as used herein in reference to polynucleotides, it is understood that the indicated elements are functionally related to each other and are also generally physically related. Thus, the term operationally linked or operationally associated, as used herein, refers to nucleotide sequences in a single nucleic acid molecule that are functionally associated. Thus, a first nucleotide sequence that is operationally linked to a second nucleotide sequence means a situation in which the first nucleotide sequence is placed in a functional relationship with the second nucleotide sequence. For example, a promoter is operationally associated with a nucleotide sequence if the promoter affects the transcription or expression of said nucleotide sequence.Those skilled in the art will appreciate that control sequences (e.g., promoter) need not be contiguous to the nucleotide sequence to which they are operatively associated, provided that the control sequences function to direct their expression. Thus, for example, untranslated but transcribed nucleic acid sequences may be present between a promoter and the nucleotide sequence, and the promoter may still be considered. Petition 870250103857, dated 11 / 13 / 2025, page 64 / 212 55 / 166 operationally linked to the nucleotide sequence.

[00146] As used herein, the term linked, in reference to polypeptides, refers to the linking of one polypeptide to another. A polypeptide may be linked to another polypeptide (at the N-terminus or the C-terminus) directly (e.g., via a peptide bond) or via a linker.

[00147] The term linker is recognized in the art and refers to a chemical group, or a molecule that links two molecules or fractions, for example, two domains of a fusion protein, such as, for example, a nucleic acid-binding polypeptide or domain and peptide marker and / or a reverse transcriptase and an affinity polypeptide that binds to the peptide marker; or a DNA endonuclease polypeptide or domain and peptide marker and / or a reverse transcriptase and an affinity polypeptide that binds to the peptide marker. A linker may consist of a single linker molecule or may comprise more than one linker molecule. In some embodiments, the linker may be an organic molecule, group, polymer, or chemical fraction, such as a bivalent organic fraction. In some embodiments, the linker may be an amino acid or a peptide. In some embodiments, the linker is a peptide.

[00148] In some embodiments, a useful peptide linker with this invention may have from about 2 to about 100 or more amino acids in length, for example, 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, 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 amino acids in length (for example, about 2 to about 40, about 2 to Petition 870250103857, dated 11 / 13 / 2025, p. 65 / 212 56 / 166 near 50, near 2 to near 60, near 4 to near 40, near 4 to near 50, near 4 to near 60, near 5 to near 40, near 5 to near 50, near 5 to near 60, near 9 to near 40, near 9 to near 50, near 9 to near 60, near 10 to near 40, near 10 to near 50, near 10 to near 60, or near 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 amino acids for approximately 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 amino acids in length (for example, about 105, 110, 115, 120, 130, 140, 150 or more amino acids in length). In some embodiments, a peptide linker may be a GS linker.

[00149] As used herein, the term linked or fused in reference to polynucleotides refers to the linking of one polynucleotide to another. In some embodiments, two or more polynucleotide molecules may be linked by a linker which may be an organic molecule, group, polymer or chemical moiety, such as a bivalent organic moiety. A polynucleotide may be linked or fused to another polynucleotide (at the 5' or 3' end) by means of a covalent or non-covalent link or bond, including, for example, Watson-Crick base pairing, or by means of one or more linking nucleotides. In some embodiments, a polynucleotide motif of a particular structure may be inserted within another polynucleotide sequence (e.g., hairpin structure extension in guide RNA). In some embodiments, the linking nucleotides may be naturally occurring nucleotides.In some forms, the linking nucleotides may be non-natural nucleotides. Petition 870250103857, dated 11 / 13 / 2025, p. 66 / 212 57 / 166

[00150] A “promoter” is a nucleotide sequence that controls or regulates the transcription of a nucleotide sequence (e.g., a coding sequence) that is operatively associated with the promoter. The coding sequence controlled or regulated by a promoter may encode a polypeptide and / or a functional RNA. Typically, a promoter refers to a nucleotide sequence that contains a binding site for RNA polymerase II and directs the initiation of transcription. In general, promoters are found 5', or upstream, relative to the start of the coding region of the corresponding coding sequence. A promoter may comprise other elements that act as regulators of gene expression; for example, a promoter region. This includes a TATA box consensus sequence and, frequently, a CAAT box consensus sequence (Breathnach and Chambon, (1981) Annu. Rev. Biochem. 50:349).In plants, the CAAT box can be replaced by the AGGA box (Messing et al., (1983) in Genetic Engineering of Plants, T. Kosuge, C. Meredith and A. Hollaender (eds.), Plenum Press, pp. 211-227).

[00151] Useful promoters with this invention may include, for example, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and / or tissue-specific promoters for use in the preparation of recombinant nucleic acid molecules, for example, synthetic nucleic acid constructs or protein-RNA complexes. These various types of promoters are known in the art.

[00152] The choice of promoter may vary depending on the temporal and spatial requirements for expression, and may also vary based on the host cell to be transformed. Promoters for many different organisms are well known in the art. Based on the extensive knowledge present in the art, the appropriate promoter may Petition 870250103857, dated 11 / 13 / 2025, page 67 / 212 58 / 166 to be selected for the specific host organism of interest. Thus, for example, much is known about promoters upstream of genes that are highly expressed constitutively in model organisms, and this knowledge can be easily accessed and implemented in other systems, as appropriate.

[00153] In some embodiments, a functional promoter in a plant can be used with the constructs of this invention. Non-limiting examples of a useful promoter for driving expression in a plant include the RubisCo small subunit 1 gene promoter (PrbcS1), the actin gene promoter (Pactin), the nitrate reductase gene promoter (Pnr), and the duplicate carbonic anhydrase 1 gene promoter (Pdca1) (see Walker et al. Plant Cell Rep. 23:727-735 (2005); Li et al. Gene 403: 132-142 (2007)). PrbcS1 and Pactin are constitutive promoters, and Pnr and Pdca1 are inducible promoters. Pnr is nitrate-induced and ammonium-repressed (Li et al. Gene 403: 132-142 (2007)), and Pdca1 is salt-induced (Li et al. Mol Biol. Rep. 37: 1143-1154 (2010)). In some embodiments, a useful promoter with this invention is the RNA polymerase II (Pol II) promoter.In some embodiments, a U6 promoter or a 7SL promoter from Zea mays may be useful with constructs of this invention. In some embodiments, the U6c promoter and / or the 7SL promoter from Zea mays may be useful for targeting the expression of a guide nucleic acid. In some embodiments, a U6c promoter, a U6i promoter, and / or a 7SL promoter from Glycine max may be useful with constructs of this invention. In some embodiments, the U6c promoter, the U6i promoter, and / or the 7SL promoter from Glycine max may be useful for targeting the expression of a guide nucleic acid.

[00154] Examples of useful constitutive promoters for plants include, but are not limited to, the cestrum virus promoter (cmp) Petition 870250103857, dated 11 / 13 / 2025, page 68 / 212 59 / 166 (U.S. Patent No. 7,166,770), the rice actin 1 promoter (Wang et al. (1992) Molecular. Cell. Biology. 12:3399-3406; as well as U.S. Patent No. 5,641,876), CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812), CaMV 19S promoter (Lawton et al. (1987) Plant Mol. Biol. 9:315-324), nos promoter (Ebert et al. (1987) Proc. Natl. Acad. Sci. USA 84:5745-5749), Adh promoter (Walker et al. (1987) Proc. Natl. Acad. Sci. USA) 84:6624-6629), sucrose synthase promoter (Yang & Russell (1990) Proc. Natl. Acad. Sci. USA 87:41444148) and the ubiquitin promoter. The constitutive ubiquitin-derived promoter accumulates in many cell types. Ubiquitin promoters have been cloned from several plant species for use in transgenic plants, for example, sunflower (Binet et al., 1991. Plant Science 79: 87-94), maize (Christensen et al., 1989. Plant Molec. Biol. 12: 619-632) and Arabidopsis (Norris et al. 1993. Plant Molec. Biol. (21:895-906).The maize ubiquitin promoter (UbiP) was developed in transgenic monocotyledon systems, and its sequence and vectors constructed for monocotyledon transformation are disclosed in patent publication EP 0 342 926. The ubiquitin promoter is suitable for the expression of the nucleotide sequences of the invention in transgenic plants, especially monocotyledons. Furthermore, the promoter expression cassettes described by McElroy et al. (Mol. Gen. Genet. 231: 150-160 (1991)) can be easily modified for the expression of the nucleotide sequences of the invention and are particularly suitable for use in monocotyledon hosts.

[00155] In some embodiments, tissue-specific / tissue-preferred promoters can be used for the expression of a heterologous polynucleotide in a plant cell. Tissue-specific or tissue-preferred expression patterns include, among others, green tissue-specific or preferred, tissue-specific or preferred Petition 870250103857, dated 11 / 13 / 2025, page 69 / 212 60 / 166 root, stem-specific or preferential, flower-specific or preferential, or pollen-specific or preferential promoters. Suitable promoters for expression in green tissue include many that regulate genes involved in photosynthesis, and many of them have been cloned from monocots and dicots. In one embodiment, a useful promoter with the invention is the maize PEPC promoter of the phosphoenol carboxylase gene (Hudspeth & Grula, Plant Molec. Biol. 12:579-589 (1989)).Non-limiting examples of tissue-specific promoters include those associated with genes encoding seed storage proteins (such as β-conglycinin, cruciferin, napin, and phaseolin), zein or oil body proteins (such as oleosin), or proteins involved in fatty acid biosynthesis (including acyl transporter protein, stearoyl-ACP desaturase, and fatty acid desaturases (fad 2-1)) and other nucleic acids expressed during embryo development (such as Bce4; see, for example, Kridl et al. (1991) Seed Sci. Res. 1:209-219; as well as EP Patent No. 255378). Tissue-specific or tissue-preferred promoters useful for the expression of the nucleotide sequences of the invention in plants, particularly maize, include, among others, those that direct expression in the root, pith, leaf, or pollen. These promoters are disclosed, for example, in WO 93 / 07278, which is incorporated herein by reference in its entirety.Other non-limiting examples of tissue-specific or tissue-preferred promoters useful with the invention are the cotton rubisco promoter disclosed in U.S. Patent 6,040,504; the rice sucrose synthase promoter disclosed in U.S. Patent 5,604,121; the root-specific promoter described by de Framond (FEBS 290:103-106 (1991); EP 0 452 269 to Ciba-Geigy); the stem-specific promoter described in U.S. Patent 5,625,136 (to Ciba-Geigy) and which directs the expression of the maize trpA gene; the amar leafroll virus promoter. Petition 870250103857, dated 11 / 13 / 2025, pp. 70 / 212 61 / 166 relo da folha do oestrum divulged no WO 01 / 73087; and specific or preferred pollen promoters, including, but not limited to, ProOsLPSW and ProOsLPSH from rice (Nguyen et al. Plant Biotechnol. Reports 9(5):297-306 (2015)), ZmSTK2_USP from maize (Wang et al. Genome 60(6):485-495 (2017)), LAT52 and LAT59 from tomato (Twell et al. Development 109(3):705-713 (1990)), Zm13 (US Patent No. 10,421,972), the PLA2-O promoter from Arabidopsis (US Patent No. 7,141,424) and / or the ZmC5 promoter from maize (PCT International Publication No. W01999 / 042587).

[00156] Additional examples of plant tissue-specific / tissue-preferred promoters include, but are not limited to, root hair-specific cis-elements (RHEs) (Kim et al. The Plant Cell 18:2958-2970 (2006)), root-specific promoters RCc3 (Jeong et al. Plant Physiol. 153:185-197 (2010)) and RB7 (US Patent No. 5459252), the lectin promoter (Lindstrom et al. (1990) Der. Genet. 11:160-167; and Vodkin (1983) Prog. Clin. Biol. Res. 138:87-98), corn alcohol dehydrogenase 1 promoter (Dennis et al. (1984) Nucleic Acids Res. 12:3983-4000), S-adenosyl-L-methionine synthetase (SAMS) (Vander Mijnsbrugge et al. (1996) Plant and Cell Physiology, 37(8):1108-1115), promoter of the maize light harvesting complex (Bansal et al. (1992) Proc. Natl. Acad. Sci. USA 89:3654-3658), promoter of the maize heat shock protein (O'Dell et al. (1985) EMBO J. 5:451-458; and Rochester et al (1986) EMBO J.5:451-458), promoter of the small subunit of pea RuBP carboxylase (Cashmore, Nuclear genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase pp. 29-39 In: Genetic Engineering of Plants (Hollaender ed., Plenum Press 1983; and Poulsen et al. (1986) Mol. Gen. Genet. 205:193-200), mannopine synthase promoter from the Ti plasmid (Langridge et al. (1989) Proc. Natl. Acad. Sci. USA 86:3219-3223), nopaline synthase promoter from the Ti plasmid (Langridge et al. (1989), su. Petition 870250103857, dated 11 / 13 / 2025, page 71 / 212 62 / 166 pra), petunia chalcone isomerase promoter (van Tunen et al. (1988) EMBO J. 7:1257-1263), bean glycine-rich protein 1 promoter (Keller et al. (1989) Genes Dev. 3:1639-1646), truncated CaMV 35S promoter (O'Dell et al. (1985) Nature 313:810-812), potato patatin promoter (Wenzler et al. (1989) Plant Mol. Biol. 13:347-354), root cell promoter (Yamamoto et al. (1990) Nucleic Acids Res. 18:7449), maize zein promoter (Kriz et al. (1987) Mol. Gen. Genet. 207:90-98; Langridge et al. (1983) Cell 34:1015-1022; Reina et al. (1990) Nucleic Acids Res. 18:6425; Reina et al. (1990) Nucleic Acids Res. 18:7449; and Wandelt et al. (1989) Nucleic Acids Res. 17:2354), globulin-1 promoter (Belanger et al. (1991) Genetics 129:863-872), α-tubulin taxi promoter (Sullivan et al. (1989) Mol. Gen. Genet. 215:431-440), PEPCase promoter (Hudspeth & Grula (1989) Plant Mol. Biol. 12:579-589), promoters associated with the R gene complex (Chandler et al. (1989) Plant Cell 1:1175-1183) and chalcone synthase promoters (Franken et al. (1991) EMBO J. 10:2605-2612).

[00157] Useful for specific seed expression is the pea vicilin promoter (Czako et al. (1992) Mol. Gen. Genet. 235:33-40; as well as the specific seed promoters disclosed in U.S. Patent No. 5,625,136. Promoters useful for expression in mature leaves are those that are switched off at the beginning of senescence, such as the Arabidopsis SAG promoter (Gan et al. (1995) Science 270:1986-1988).

[00158] In addition, functional promoters in chloroplasts can be used. Non-limiting examples of such promoters include the 9 5' UTR gene of bacteriophage T3 and other promoters disclosed in U.S. Patent No. 7,579,516. Other useful promoters with the invention include, among others, the promoter of the small subunit RuBP carboxylase S-E9 and the promoter of the Kunitz trypsin inhibitor gene (Kti3). Petition 870250103857, dated 11 / 13 / 2025, page 72 / 212 63 / 166

[00159] Additional regulatory elements useful with this invention include, among others, introns, enhancers, termination sequences and / or untranslated 5' and 3' regions.

[00160] A useful intron with this invention may be an intron identified and isolated from a plant and then inserted into an expression cassette for use in plant transformation. As would be understood by those skilled in the art, introns may comprise the sequences required for autoexcision and are incorporated into nucleic acid constructs / in-frame expression cassettes. An intron may be used as a spacer to separate multiple protein-coding sequences in a nucleic acid construct, or an intron may be used within a protein-coding sequence to, for example, stabilize mRNA. If used within a protein-coding sequence, they will be inserted in-frame with the excision sites included. Introns may also be associated with promoters to enhance or modify expression.As an example, a useful promoter / intron combination with this invention includes, among others, that of the Ubi1 promoter and intron from maize (see, for example, SEQ ID NO:21 and SEQ ID NO:22).

[00161] Non-limiting examples of useful introns with the present invention include introns of the ADHI gene (e.g., introns 1, 2 and 6 of Adh1-S), the ubiquitin gene (Ubi1), the RuBisCO small subunit gene (rbcS), the RuBisCO large subunit gene (rbcL), the actin gene (e.g., actin-1 intron), the pyruvate dehydrogenase kinase (pdk) subunit gene, the nitrate reductase (nr) gene, the duplicated carbonic anhydrase 1 (Tdca1) gene, the psbA gene, the atpA gene, or any combination thereof.

[00162] In some embodiments, a polynucleotide and / or nucleic acid construct of the invention may be an ex cassette. Petition 870250103857, dated 11 / 13 / 2025, p. 73 / 212 64 / 166 pressure or may be contained within an expression cassette. As used herein, expression cassette means a recombinant nucleic acid molecule comprising, for example, one or more polynucleotides of the invention (for example, a polynucleotide encoding a sequence-specific nucleic acid binding domain, a polynucleotide encoding a protein or deaminase domain, a polynucleotide encoding a protein or reverse transcriptase domain, a polynucleotide encoding a 5'-3' exonuclease polypeptide or domain, a guide nucleic acid and / or reverse transcriptase (RT) template), wherein the polynucleotides are operationally associated with one or more control sequences (for example, a promoter, terminator and the like).Thus, in some embodiments, one or more expression cassettes may be provided, which are designed to express, for example, a nucleic acid construct of the invention (for example, a polynucleotide encoding a sequence-specific nucleic acid binding domain, a polynucleotide encoding a nuclease polypeptide / domain, a polynucleotide encoding a deaminase protein / domain, a polynucleotide encoding a reverse transcriptase protein / domain, a polynucleotide encoding a 5'-3' exonuclease polypeptide / domain, a polynucleotide encoding a peptide marker and / or a polynucleotide encoding an affinity polypeptide and the like, or comprising a guide nucleic acid, an extended guide nucleic acid and / or RT template and the like).When an expression cassette of the present invention comprises more than one polynucleotide, the polynucleotides may be operationally linked to a single promoter that directs the expression of all polynucleotides, or the polynucleotides may be operationally linked to one or more separate promoters (for example, three polynucleotides may be directed by one, two, or three promoters). Petition 870250103857, dated 11 / 13 / 2025, page 74 / 212 65 / 166 any combination). When two or more separate promoters are used, the promoters may be the same promoter or they may be different promoters. Thus, a polynucleotide encoding a sequence-specific nucleic acid binding domain, a polynucleotide encoding a protein / nuclease domain, a polynucleotide encoding a protein / CRISPR-Cas effector domain, a polynucleotide encoding a protein / deaminase domain, a polynucleotide encoding a polypeptide / reverse transcriptase domain (e.g., RNA-dependent DNA polymerase) and / or a polynucleotide encoding a 5'-3' exonuclease polypeptide / domain, a guide nucleic acid, an extended guide nucleic acid and / or RT template when contained in a single expression cassette may each be operationally linked to a single promoter, or separate promoters in any combination.

[00163] An expression cassette comprising a nucleic acid construct of the invention may be chimeric, meaning that at least one (e.g., one or more) of its components is heterologous with respect to at least one of its other components (e.g., a host organism promoter operationally linked to a polynucleotide of interest to be expressed in the host organism, wherein the polynucleotide of interest is from an organism other than the host or is not normally found in association with that promoter). An expression cassette may also be one that occurs naturally but has been obtained in a recombinant form useful for heterologous expression.

[00164] An expression cassette may optionally include a transcriptional and / or translational termination region (i.e., termination region) and / or an enhancer region that is functional in the selected host cell. A variety of transcriptional terminators and enhancers are known in the art and are available. Petition 870250103857, dated 11 / 13 / 2025, page 75 / 212 66 / 166 suitable for use in expression cassettes. Transcriptional terminators are responsible for terminating transcription and for the correct polyadenylation of mRNA. A termination region and / or enhancer region may be native to the transcriptional initiation region, may be native to, for example, a gene encoding a sequence-specific nucleic acid-binding protein, a gene encoding a nuclease, a gene encoding a reverse transcriptase, a gene encoding a deaminase and the like, or may be native to a host cell, or may be native to another source (e.g., foreign or heterologous to, for example, a promoter, a gene encoding a sequence-specific nucleic acid-binding protein, a gene encoding a nuclease, a gene encoding a reverse transcriptase, a gene encoding a deaminase and the like, or the host cell, or any combination thereof).

[00165] An expression cassette of the invention may also include a polynucleotide encoding a selectable marker, which can be used to select a transformed host cell. As used herein, selectable marker means a polynucleotide sequence that, when expressed, confers a distinct phenotype to the host cell expressing the marker and thus allows such transformed cells to be distinguished from those that do not possess the marker. This polynucleotide sequence may encode a selectable or traceable marker, depending on whether the marker confers a characteristic that can be selected by chemical means, such as using a selective agent (e.g., an antibiotic and the like), or whether the marker is simply a characteristic that can be identified by observation or testing, such as by screening (e.g., fluorescence).Many examples of suitable selectable markers are known in the art and can be used. Petition 870250103857, dated 11 / 13 / 2025, p. 76 / 212. 67 / 166 of the expression cassettes described in this document.

[00166] In addition to expression cassettes, the nucleic acid molecules / constructs and polynucleotide sequences described in this document may be used in connection with vectors. The term vector refers to a composition for transferring, delivering, or introducing a nucleic acid (or nucleic acids) into a cell. A vector comprises a nucleic acid construct (e.g., expression cassette(s)) comprising the nucleotide sequence(s) to be transferred, delivered, or introduced. Vectors for use in the transformation of host organisms are well known in the art. Non-limiting examples of general classes of vectors include viral vectors, plasmid vectors, phage vectors, phagemid vectors, cosmid vectors, fosmid vectors, bacteriophages, artificial chromosomes, minicircles, or binary vectors of Agrobacterium in linear or circular form of double or single strands, which may or may not be self-transmissible or mobilizable.In some embodiments, a viral vector may include, among others, a retroviral, lentiviral, adenoviral, adeno-associated, or herpes simplex viral vector. A vector as defined in this document may transform a prokaryotic or eukaryotic host by integration into the cellular genome or exist extrachromosomally (e.g., a self-replicating plasmid with an origin of replication). Additionally, bifunctional vectors (shuttle vectors) are included, meaning a DNA carrier capable, naturally or by design, of replicating in two different host organisms, which may be selected from actinomycetes and related species, bacteria, and eukaryotes (e.g., higher plant, mammalian, yeast, or fungal cells). In some embodiments, the nucleic acid in the vector is under the control of, and operationally linked to, an appropriate promoter or other regulatory elements for transcription into a host cell. The vector may be... Petition 870250103857, dated 11 / 13 / 2025, page 77 / 212 68 / 166 a bifunctional expression vector that functions in multiple hosts. In the case of genomic DNA, it may contain its own promoter and / or other regulatory elements, and in the case of cDNA, it may be under the control of an appropriate promoter and / or other regulatory elements for expression in the host cell. Consequently, a nucleic acid or polynucleotide of this invention and / or expression cassettes comprising it may be comprised in vectors as described in this document and as known in the art.

[00167] As used herein, contact, contacting, contacted, and grammatical variations thereof, refer to the bringing together of the components of a desired reaction under suitable conditions to accomplish the desired reaction (e.g., transformation, transcriptional control, genome editing, cleavage, and / or splitting). As an example, a target nucleic acid may be brought into contact with a sequence-specific nucleic acid-binding protein (e.g., 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) and a deaminase or a nucleic acid construct encoding the same, under conditions where the sequence-specific nucleic acid-binding protein,Reverse transcriptase and / or deaminase are expressed and the sequence-specific nucleic acid-binding protein binds to the target nucleic acid, and the reverse transcriptase and / or deaminase may be fused to the sequence-specific nucleic acid-binding protein or recruited to the sequence-specific nucleic acid-binding protein (for example, by means of a peptide tag fused to the sequence-specific nucleic acid-binding protein and an affinity tag fused to it). Petition 870250103857, dated 11 / 13 / 2025, pp. 78 / 212 69 / 166 to reverse transcriptase and / or deaminase) and thus, the deaminase and / or reverse transcriptase is positioned in the vicinity of the target nucleic acid, thereby modifying the target nucleic acid. Other methods for recruiting reverse transcriptase and / or deaminase can be used to remove markers from other protein-protein interactions, and RNA-protein interactions and chemical interactions can also be used for protein-protein and protein-nucleic acid recruitment.

[00168] As used herein, modifying or modifying with reference to a target nucleic acid includes editing (e.g., mutation), covalent modification, nucleic acid / nucleotide base exchange / substitution, deletion, cleavage, cutting, and / or alteration of the transcriptional control of a target nucleic acid. In some embodiments, a modification may include one or more single base alterations (SNPs) of any type.

[00169] To introduce, introduces, introduced (and grammatical variations thereof) in the context of a polynucleotide of interest means to present a nucleotide sequence of interest (e.g., polynucleotide, RT template, nucleic acid construct and / or guide nucleic acid) to a plant, a part thereof or a cell thereof, in such a way that the nucleotide sequence gains access to the interior of a cell.

[00170] The terms transformation or transfection may be used interchangeably and, as used in this document, refer to the introduction of a heterologous nucleic acid into a cell. The transformation of a cell may be stable or transient. Thus, in some embodiments, a host cell or host organism (e.g., a plant) may be stably transformed with a polynucleotide / nucleic acid molecule of the invention. In some embodiments, a host cell or organism Petition 870250103857, dated 11 / 13 / 2025, pp. 79 / 212 The 70 / 166 host can be transiently transformed with a polynucleotide / nucleic acid molecule of the invention.

[00171] Transient transformation in the context of a polynucleotide means that a polynucleotide is introduced into the cell and does not integrate into the cell's genome.

[00172] By stable introduction or stably introduced in the context of a polynucleotide introduced into a cell, it is understood that the introduced polynucleotide is stably incorporated into the cell's genome and thus the cell is stably transformed with the polynucleotide.

[00173] Stable transformation or stably transformed, as used herein, means that a nucleic acid molecule is introduced into a cell and integrates into the cell's genome. In this way, the integrated nucleic acid molecule is capable of being inherited by its progeny, more particularly, by the progeny of multiple successive generations. Genome, as used herein, includes the nuclear and plastid genome and therefore includes the integration of nucleic acid, for example, into the chloroplast or mitochondrial genome. Stable transformation, as used herein, may also refer to a transgene that is maintained extrachromosomally, for example, as a minichromosome or a plasmid.

[00174] Transient transformation can be detected, for example, by 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, for example, by a Southern blot hybridization assay of the cell's genomic DNA with nucleic acid sequences that specifically hybridize with a nucleotide sequence of a transgene introduced into an organism (e.g., a plant). Stable transformation of a cell Petition 870250103857, dated 11 / 13 / 2025, pp. 80 / 212 71 / 166 Squid can be detected, for example, by a Northern blot hybridization assay of the cell's RNA with nucleic acid sequences that specifically hybridize with a nucleotide sequence of a transgene introduced into a host organism. Stable transformation of a cell can also be detected, for example, by a polymerase chain reaction (PCR) or other amplification reactions well known in the art, employing specific primer sequences that hybridize with target sequences 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.

[00175] Consequently, in some embodiments, nucleotide sequences, polynucleotides, nucleic acid constructs and / or expression cassettes of the invention may be transiently expressed and / or may be stably incorporated into the genome of the host organism. Thus, in some embodiments, a nucleic acid construct of the invention (for example, one or more expression cassettes comprising polynucleotides for editing as described in this document) may be transiently introduced into a cell with a guide nucleic acid and, as such, no DNA is retained in the cell.

[00176] A nucleic acid construct of the invention can be introduced into a plant cell by any method known to those skilled in the art. Non-limiting examples of transformation methods include transformation via nucleic acid administration mediated by bacteria (e.g., via Agrobacteria), nucleic acid administration mediated by viruses, nucleic acid administration mediated by silicon carbide or nucleic acid whisker, nucleic acid administration mediated by liposomes, microin Petition 870250103857, dated 11 / 13 / 2025, p. 81 / 212 72 / 166 injection, 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 nucleic acid into the plant cell, including any combination thereof. Procedures for transforming eukaryotic and prokaryotic organisms are well known and routine in the field 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 guidelines for various plant transformation methods known in the art include Miki et al. (Procedures for Introducing Foreign DNA into Plants in Methods in Molecular Biology and Plant Biotechnology, Glick, BR and Thompson, JE, Eds. (CRC Press, Inc.), Boca Raton, 1993), pages 67-88) and RakowoczyTrojanowska (Cell. Mol. Biol. Lett. 7:849-858 (2002)).

[00177] In some embodiments of the invention, the transformation of a cell may comprise nuclear transformation. In other embodiments, the transformation of a cell may comprise plastid transformation (e.g., chloroplast transformation). In other embodiments, the nucleic acids of the invention may be introduced into a cell by means of conventional breeding techniques. In some embodiments, one or more polynucleotides, expression cassettes and / or vectors may be introduced into a plant cell by means of Agrobacterium transformation.

[00178] A polynucleotide, therefore, can be introduced into a plant, plant part, or plant cell in several ways well known in the art. The methods of the invention do not depend on a specific method for introducing one or more nucleotide sequences into a plant, only on their having access to the interior of the cell. Petition 870250103857, dated 11 / 13 / 2025, page 82 / 212 73 / 166 la. When more than one polynucleotide needs to be introduced, they can be assembled as part of a single nucleic acid construct, or as separate nucleic acid constructs, and can be located in the same nucleic acid construct or in different nucleic acid constructs. Consequently, the polynucleotide can be introduced into the cell of interest in a single transformation event, or in separate transformation events, or, alternatively, a polynucleotide can be incorporated into a plant as part of a breeding protocol.

[00179] In agricultural and horticultural settings, thorns can cause significant problems for those who handle the plants. Thorns interfere, for example, with the propagation, care, and harvesting of these crops and, as a result, can lead to increased costs. As an example, the stems of wild blackberry and other Rubus species are covered from bottom to top with varying densities of thorns. These thorns are not derived from the epidermis, but rather from the vascular tissue and are more correctly called spines. In addition to interfering with plant propagation, plant care, and fruit harvesting, the presence of thorns can be a risk if the consumer inadvertently eats a thorn.

[00180] Today, there are four sources of thornless traits available to blackberry growers. One of them is the recessive 's' gene used by the John Innes program in the UK to develop 'Merton Thornless', which is still the source of thornless traits in modern tetraploid blackberry cultivars. The use of 'Merton Thornless' has been very important for the advancement of thornless traits, particularly in erect and semi-erect genotypes. The 's' gene is a recessive trait. Two advantages of this gene are the complete absence of thorns and the ability to track seedlings. Petition 870250103857, dated 11 / 13 / 2025, page 83 / 212 74 / 166 regarding the absence of glandular trichomes. Disadvantages include a segregation rate of 35:1 in tetraploids and the linking of this characteristic to undesirable traits such as lack of winter hardiness, acidic fruits, and late harvest.

[00181] Another source of thornless blackberry is the dominant 'Sf' gene from 'Austin Thornless'. The advantage of 'Sf' is its genetic dominance, which has provided a thornless source at 6x and higher ploidy levels ('Austin Thornless' is an octoploid). 'Sf' has been important in breeding trailing varieties of blackberries. However, several detrimental traits are associated with this location, including slow growth habit and susceptibility to mildew. Furthermore, plants derived from this thornless source may have thorns in the basal 0.3 m of the cane; these same canes are thornless beyond this point and are commercially thornless, as the fruit only grows in the thornless area of ​​the cane.

[00182] The dominant locus 'Sfte' is another source of spinelessness and comes from non-chimeric 'Thornless Evergreen' plants. This locus is not used in breeding as it reproduces regularly in the field and is also associated with many undesirable traits. A fourth source of spinelessness available for breeding includes the semi-dominant 'Sfl' gene from 'Lincoln Logan'. This allele was originally identified as a chimera of 'Loganberry'. A tissue culture technique in which a 'Loganberry' type clone (L654) was used resulted in a spontaneous embryo from callus tissue. 'Lincoln Logan' was released from this effort and was then used in the New Zealand and USDA-ARS Oregon breeding programs. Early cultivars with the 'Sfl' source are not yet commercially used; however, there are extensive fertility problems associated with 'Sfl' and it also appears that two separate loci may be Petition 870250103857, dated 11 / 13 / 2025, page 84 / 212 75 / 166 are needed to produce thornless 'Sfl'.

[00183] Blackberry varieties are propagated clonally. Currently, if breeders have a thorny blackberry variety, it is not possible to remove the thorns without laborious crosses and backcrosses to introduce one of the four thornless alleles mentioned. Making a thornless blackberry plant in one generation would be a drastic advance for blackberry genetic improvement. Such an advance would be useful in other Rosaceae plants, including other Rubus spp., as well as Rosa spp., Hulthemia spp., Hesperrhodos spp., or Platyrhodon spp.

[00184] The present invention is directed to modifying genes that contribute to the development of thorns and prickles in order to improve the handling of these plants and reduce the costs associated with working with these plants compared to varieties that include thorns and / or prickles. Thus, a useful editing strategy for this invention may include generating a mutation in one or more of a functional WOX gene in a Rosaceae plant. In some embodiments, one or more mutations (optionally, an unnatural mutation) may be generated in a functional WOX gene of a plant. Mutations that may be useful for producing Rosaceae plants with a reduced number of thorns and / or prickles, thorns and / or prickles that have modified architecture, thorns and / or prickles that are reduced in size and / or without thorns and / or prickles include, for example, substitutions, deletions and / or insertions.In some respects, a mutation generated by editing technology can be a point mutation. In some modalities, the mutation can be an out-of-frame insertion or an out-of-frame deletion, optionally where the out-of-frame insertion or out-of-frame deletion produces / results in a premature stop codon. In some modalities, a mutation in... Petition 870250103857, dated 11 / 13 / 2025, page 85 / 212 76 / 166 one or more of a functional WOX gene as described herein results in the production of a modified WOX transcription factor, optionally wherein the modified WOX transcription factor comprises a C-terminal truncation, optionally a truncation of about the last 200-320 consecutive amino acid residues (about 200, 205, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 311, 313, 314, 315, 316, 317, 318, 319, or 320 consecutive amino acid residues, or any value or range therein) compared to a WOX transcription factor produced by an unmodified endogenous functional WOX gene. In some embodiments, a mutation in one or more of a functional WOX gene as described herein results in the production of a reduced amount or no encoded polypeptide, or in the encoded WOX transcription factor being undetectable.

[00185] In some embodiments, a Rosaceae plant or part thereof is provided comprising at least one mutation in at least one (e.g., one or more) endogenous functional homebox-related WUSCHEL (WUS) (WOX) gene encoding a WOX transcription factor, optionally wherein at least one mutation may be a non-natural mutation. At least one mutation in at least one endogenous functional WOX gene provides a mutation in the WOX gene that may be a non-functional WOX gene that may encode a non-functional WOX transcription factor. In some embodiments, the encoded WOX transcription factor regulates the production of thorns and / or prickles. In some embodiments, the endogenous functional WOX gene is specifically expressed in tissues that give rise to trichomes, thorns, and / or prickles. In some embodiments, the endogenous functional WOX gene is specifically expressed in vascular tissue, epidermal tissue, and / or cells or Petition 870250103857, dated 11 / 13 / 2025, page 86 / 212 77 / 166 tissues that give rise to a thorn or spine. In some embodiments, the endogenous functional WOX gene is an endogenous functional WOX1 gene and / or an endogenous functional WOX23 gene, and the encoded WOX transcription factor is a WOX1 polypeptide or a WOX23 polypeptide, respectively. In some embodiments, a Rosaceae plant or part of a plant may comprise at least one mutation in two or more endogenous functional WOX genes.

[00186] In some embodiments, a mutation in an endogenous functional WOX gene may be located in the first or second exon of the gene, optionally where the mutation is located in the first and / or second exon of the WOX1 gene and / or in the second exon of the WOX23 gene. In some embodiments, a mutation may be an out-of-frame insertion or an out-of-frame deletion, optionally where the out-of-frame insertion or out-of-frame deletion produces / results in a premature stop codon, optionally resulting in a C-terminal truncation of the encoded transcription factor (e.g., a truncated WOX transcription factor). In some embodiments, a mutation in the endogenous functional WOX gene may result in a WOX gene mutation that produces a truncated WOX transcription factor or does not produce any detectable WOX transcription factor.In some embodiments, a C-terminal truncation may comprise a deletion of one amino acid residue for approximately 320 consecutive amino acid residues of the encoded WOX transcription factor. In some embodiments, a mutation of a functional WOX gene in a Rosaceae plant may result in a dominant mutation, a recessive mutation, a semi-dominant mutation, or a hypomorphic mutation. In some embodiments, a Rosaceae plant or part thereof comprising at least one mutation in an endogenous functional gene. Petition 870250103857, dated 11 / 13 / 2025, p. 87 / 212 78 / 166 WOX exhibits a phenotype of a reduced number of spines and / or prickles, spines and / or prickles with modified architecture, spines and / or prickles of reduced size, and / or absence of spines and / or prickles when compared to a control Rosaceae plant lacking at least one mutation.

[00187] In some embodiments, a mutant WOX gene contained in a Rosaceae plant cell may have at least 90% sequence identity with any of the mutant WOX genes (e.g., WOX1, WOX23) described herein.

[00188] In some embodiments, a Rosaceae plant cell is provided, the Rosaceae plant cell comprising an editing system, the editing system comprising: (a) a CRISPR-Cas effector protein; and (b) a guide nucleic acid (e.g., gRNA, gDNA, crRNA, crDNA, sgRNA, sgDNA) comprising a spacer sequence with complementarity to an endogenous functional target gene encoding a WUSCHEL-related Homeobox (WOX) transcription factor in the Rosaceae plant cell. The editing system can be used to generate a mutation in the endogenous functional target gene encoding a WOX transcription factor.In some embodiments, the endogenous functional target gene is a WUSCHEL-related (WUS) Homebox-related endogenous functional gene (WOX) (e.g., one or more endogenous functional WOX genes), optionally an endogenous functional WOX1 gene and / or an endogenous functional WOX23 gene, and the WOX transcription factor is a WOX1 transcription factor or a WOX23 transcription factor. In some embodiments, the encoded WOX transcription factor regulates the production of spines and / or prickles. In some embodiments, the endogenous functional WOX gene is specifically expressed in tissues that give rise to trichomes, spines, and / or prickles. In some embodiments, the endogenous functional WOX gene is specifically expressed in... Petition 870250103857, dated 11 / 13 / 2025, pp. 88 / 212 79 / 166 vascular tissue and / or epidermal tissue. In some embodiments, the endogenous functional target gene WOX: (a) comprises a nucleotide sequence with at least 80% sequence identity (e.g., at least approximately 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity) with any of the SEQ ID NOs: 69, 70, 75, 76, 128, 129 or 162173; (b) comprises a region with at least 80% sequence identity with a nucleotide sequence of any of the following SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134; (c) encodes an amino acid sequence with at least 80% sequence identity with any of the following SEQ ID NOs: 71, 77, 130, or 174-182;and / or (d) encodes a region of an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 136, or SEQ ID NO: 137, optionally where the sequence identity of (a), (b), (c), and / or (d) may be at least 85%, or at least 90%, or may be at least 95%, optionally the sequence identity may be 100%. In some embodiments, a guide nucleic acid of an editing system may comprise the nucleotide sequence (a spacer sequence, for example, one or more spacers) of any of the SEQ ID NOs: 138-142, or reverse complement, or any combination thereof. In some embodiments, a mutation may be an unnatural mutation.

[00189] A mutation in a functional WOX gene of a Rosaceae plant, plant part, or Rosaceae plant cell useful for this invention may be any type of mutation, including a base substitution, a base deletion, a base insertion, and / or an inversion. In some embodiments, the mutation may be Petition 870250103857, dated 11 / 13 / 2025, pp. 89 / 212 80 / 166 an unnatural mutation. In some forms, the mutation may involve a base substitution for an A, a T, a G, or a C. In some forms, the mutation may be a deletion or insertion (e.g., of at least one base pair (e.g., 1 base pair for about 100 base pairs; e.g., 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, 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 or 100 consecutive base pairs, or any interval or value therein; optionally 1 to about 50 consecutive base pairs, 1 to about 30 consecutive base pairs, 1 to about 15 consecutive base pairs. In some embodiments, at least one mutation may be an out-of-frame insertion or an out-of-frame deletion, optionally where the mutation is an unnatural mutation. In some embodiments, the deletion or insertion (e.g., out-of-frame insertion or out-of-frame deletion) may be located in the first and / or second exon of the functional WOX gene, optionally located in the first and / or second exon of the functional WOX1 gene and / or in the second exon of the functional WOX23 gene.In some forms, a deletion or insertion (e.g., out-of-frame insertion or out-of-frame deletion) can result in a mutated WOX gene that produces a truncated WOX transcription factor polypeptide or a reduced amount or no detectable WOX transcription factor polypeptide.

[00190] In some embodiments, out-of-frame deletion or out-of-frame insertion may result in a WOX gene encoding a truncated polypeptide, optionally a polypeptide with a C-terminal truncation resulting from a premature stop codon. Petition 870250103857, dated 11 / 13 / 2025, pp. 90 / 212 81 / 166 generated by deletion or insertion. In some embodiments, C-terminal truncation is a deletion of approximately 200–320 consecutive amino acid residues (approximately 200, 205, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 311, 313, 314, 315, 316, 317, 318, 319, or 320 consecutive amino acid residues, or any value or range thereof). in it) compared to a WOX transcription factor produced by an unmodified endogenous WOX gene.

[00191] In some embodiments, a useful mutation with this invention may be a dominant mutation, a semi-dominant mutation, a hypomorphic mutation, or a recessive mutation, optionally where the mutation is an unnatural mutation.

[00192] In some embodiments, a mutation in a functional endogenous WOX gene can result in a mutated WOX nucleic acid with at least 90% sequence identity to any of the mutated WOX genes (e.g., WOX1, WOX23) described herein. The mutant WOX gene may be a non-functional endogenous WOX gene and / or may encode a non-functional WOX transcription factor.

[00193] A species of Rosaceae plant that may be modified as described herein may include, but is not limited to, Rosa spp., Hulthemia spp., a Hesperrhodos spp., a Platyrhodon spp., or a Rubus spp. In some embodiments, the Rubus spp. may be blackberry or black raspberry, optionally, wherein the Rubus spp. plant is Rubus allegheniensis (Allegheny blackberry), Rubus armeniacus (Himalayan blackberry), Rubus canadensis (Canadian blackberry), Rubus fruticosus agg. (European blackberry), Rubus laciniatus (perennial cut-leaf blackberry), Rubus pensilvanicus (Pennsylvanian blackberry), Rubus trifidus (Japanese blackberry), Rubus ursinus (wild right blackberry), Rubus hybrid (e.g., progeny resulting from hybridization). Petition 870250103857, dated 11 / 13 / 2025, pages 91 / 212 82 / 166 The combination of two or more of the above genes in Rubus species), Rubus leucodermis (white-skinned raspberry or western black raspberry) or Rubus occidentalis (black raspberry). In some embodiments, Rubus spp. may be a raspberry plant (red raspberry), including, but not limited to, Rubus crataegifolius (Asian raspberry), Rubus gunnianus (Tasmanian alpine raspberry), Rubus idaeus (red raspberry or European red raspberry), Rubus parvifolius (Australian native raspberry), Rubus phoenicolasius (wine raspberry or wine-blackberry), Rubus rosifolius (Mauritius raspberry), Rubus strigosus (American red raspberry) (syn. R. idaeus var. strigosus), or Rubus ellipticus (Himalayan yellow raspberry). In some embodiments, Rosa spp. It is a rose plant and / or Rosa spp. is Rosa chinensis.

[00194] The types of editing tools that can be used to generate these and other mutations in a functional Rosaceae WOX gene include any base editors or cutters, which are guided to a target location using spacers with at least 80% (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, or any range or value contained therein), complementarity to a portion or region of a functional WOX gene (e.g., one or more of a functional WOX gene, e.g., a functional WOX1 gene and / or a functional WOX23 gene) as described herein.

[00195] In some embodiments, a WOX gene mutation is within a portion or region of the endogenous WOX gene with at least 80% sequence identity (e.g., at least approximately 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity) with any of the nucleotide sequences of SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally SEQ ID NOs: 81, Petition 870250103857, dated 11 / 13 / 2025, pp. 92-212 83 / 166 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134, optionally where the sequence identity may be at least 85%, or at least 90%, or may be at least 95%, optionally the sequence identity may be 100%.

[00196] A functional endogenous WUSCHEL-related Homebox gene (WOX) useful with this invention (e.g., an endogenous target gene) encodes a functional polypeptide of the WOX transcription factor and includes a functional endogenous WOX1 gene and a functional endogenous WOX23 gene, which encode a WOX1 transcription factor or a WOX23 transcription factor, respectively. A useful endogenous WOX gene with this invention is specifically expressed in tissues that give rise to trichomes, spines, and / or prickles, for example, specifically expressed in vascular tissue, epidermal tissue, and / or cells or tissues that give rise to spines and / or prickles in a plant or part thereof, and the encoded WOX transcription factor regulates the production of spines and / or prickles.

[00197] In some embodiments, an endogenous WOX gene (e.g., endogenous target gene) (1) may comprise a nucleic acid sequence with at least 80% sequence identity (e.g., at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity) with one or more of SEQ ID NOs: 69, 70, 75, 76, 128, 129 and 162-173; (b) comprises a region with at least 80% sequence identity with a nucleotide sequence of any of the following SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134; (c) encodes an amino acid sequence with at least 80% sequence identity with one or more of the following SEQ ID NOs: 71, 77, Petition 870250103857, dated 11 / 13 / 2025, pp. 93 / 212 84 / 166 130, and 174-182; and / or (d) encodes a region of an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 126, 127, 136 or 137, optionally where the sequence identity of (a), (b), (c) and / or (d) may be at least 85%, or at least 90%, or may be at least 95%, optionally the sequence identity may be 100%.

[00198] Examples of functional endogenous WOX genes and encoded WOX transcription factor polypeptides useful with this invention, as well as target regions for editing and examples of edited WOX genes and their respective encoded polypeptides are provided in Table 1. Table 1. Endogenous WOX gene Genomic SEQ ID NOs Coding and polypeptide SEQ ID NOs Genomic regions SEQ ID NOs SEQ ID NOs of polypeptide regions WOX1 69 70, 71 Exon 1 78-90 Exon 2 91-109 126, 127 WOX23 75 76, 77 Exon 2 104, 105, 109-125 127 WOX1 128 129, 130 Exon 2 95-97, 104, 105, 111113, 118-120, 124, 125, 131-135 136, 137

[00199] In some embodiments, a Rosaceae plant comprising at least one (e.g., one or more, e.g., 1, 2, 3, 4, 5 or more) mutation in an endogenous functional WOX gene (in at least one endogenous functional WOX gene, e.g., in one or more functional WOX genes, e.g., functional WOX1, and / or functional WOX23) exhibits a reduced number of spines and / or prickles, spines and / or prickles that have a modified architecture, spines and / or prickles that are reduced in size and / or without spines and / or prickles (is devoid of spines and / or prickles) when compared to Petition 870250103857, dated 11 / 13 / 2025, pp. 94 / 212 85 / 166 a control Rosaceae plant lacking at least one mutation (e.g., an isogenic plant (e.g., unedited wild plant or a null segregant).

[00200] In some embodiments, a Rosaceae plant can be regenerated from a Rosaceae plant part and / or plant cell of the invention comprising a mutation in one or more of an endogenous functional WOX genes (an endogenous functional WOX1 gene and / or an endogenous functional WOX23 gene) as described herein, wherein the regenerated Rosaceae plant comprises the mutation in one or more endogenous functional WOX genes and a phenotype of reduced number of spines and / or prickles, spines and / or prickles that have a modified architecture, spines and / or prickles that are reduced in size and / or no spines and / or prickles when compared to a control Rosaceae plant devoid of at least one mutation.

[00201] In some embodiments, a Rosaceae plant cell is provided, the Rosaceae plant cell comprising at least one (e.g., one or more) mutation (optionally an unnatural mutation) within an endogenous functional gene WOX, wherein the at least one mutation is a base substitution, a base insertion, or a base deletion that is introduced using an editing system comprising a nucleic acid binding domain that binds to a target site within the endogenous functional gene WOX, wherein the endogenous functional gene WOX is specifically expressed in tissues that give rise to trichomes, spines, and / or prickles, optionally where the endogenous functional gene WOX is specifically expressed in vascular tissue, epidermal tissue, and / or cells or tissues that give rise to spines and / or prickles in a plant or part thereof. In some embodiments, the endogenous functional gene WOX: (a) comprises a nuclear sequence Petition 870250103857, dated 11 / 13 / 2025, pp. 95-212 86 / 166 tidia with at least 80% sequence identity (e.g., at least approximately 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity) with any of the following SEQ ID numbers: 69, 70, 75, 76, 128, 129 or 162-173; (b) comprises a region with at least 80% sequence identity with a nucleotide sequence of any of the following SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134; (c) encodes an amino acid sequence with at least 80% sequence identity with any of the following SEQ ID NOs: 71, 130, or 174-182;and / or (d) encodes a region of an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 136 or SEQ ID NO: 137, optionally where the sequence identity of (a), (b), (c) and / or (d) may be at least 85%, or at least 90%, or may be at least 95%, optionally the sequence identity may be 100%.

[00202] In some embodiments, base substitution, base insertion, or base deletion results, for example, in a premature stop codon. In some embodiments, base substitution, base insertion, or base deletion results, for example, in a truncated WOX transcription factor, optionally a C-terminal truncation. In some embodiments, at least one mutation is a point mutation. In some embodiments, at least one mutation within the functional WOX gene is an insertion and / or a deletion, optionally where at least one mutation is an out-of-frame insertion or an out-of-frame deletion. In some embodiments, the endogenous functional WOX gene is an endogenous functional WOX1 gene or an endogenous functional WOX23 gene. Petition 870250103857, dated 11 / 13 / 2025, pages 96 / 212 87 / 166

[00203] In some embodiments, a target site within a WOX gene of a Rosaceae plant cell may be within a region or portion of the endogenous WOX gene, the region having at least 80% sequence identity (e.g., at least approximately 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity) with any of the nucleotide sequences SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134, optionally where the sequence identity may be at least 85%, or at least 90%, or may be at least 95%, optionally the sequence identity may be 100%.In some embodiments, the target site within the WOX gene is located within a region of the endogenous WOX gene that encodes an amino acid sequence with at least 80% sequence identity to any of the SEQ ID NOs: 126, 127, 136, and / or 137.

[00204] In some embodiments, a mutation in a functional endogenous WOX gene may result in a mutated WOX nucleic acid with at least 90% sequence identity with any of the mutated WOX genes (e.g., WOX1, WOX23) having a nucleotide sequence of SEQ ID NOs: 143, 144, 145, 147, 149, 151, 153 and / or 155 and / or a mutated WOX polypeptide comprising an amino acid sequence with at least 90% sequence identity with any of the SEQ ID NOs: 146, 148, 150, 152, 154 and / or 156.

[00205] In some embodiments, the editing system further comprises a nuclease, the nucleic acid binding domain binds to a target site within a sequence with at least 80% sequence identity (e.g., at least approximately 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or Petition 870250103857, dated 11 / 13 / 2025, pp. 97 / 212 88 / 166 100% sequence identity) to any of the SEQ ID NOs: 69, 70, 75, 76, 128, 129, or 162-173 and / or to a sequence with at least 80% sequence identity to a sequence from any of the SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125, or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125, or 134, optionally where the sequence identity may be at least 85%, or at least 90%, or it may be at least 95%, optionally the sequence identity may be 100%, and at least one mutation within a functional WOX gene is produced after cleavage by nuclease, optionally where at least one mutation is an unnatural mutation. In some embodiments, at least one mutation may result in a hypomorphic mutation, a dominant mutation, a semi-dominant mutation, or a recessive mutation.

[00206] In some embodiments, at least one mutation within the functional WOX gene is an insertion and / or a deletion, optionally at least one mutation is an out-of-frame insertion or an out-of-frame deletion. In some embodiments, at least one mutation may be an insertion and / or a deletion that results in a premature stop codon, optionally resulting in a WOX transcription factor with a C-terminal truncation, optionally where the C-terminal truncation may be the deletion of an amino acid residue for about 320 consecutive amino acid residues, optionally 200-320 consecutive amino acid residues of the encoded WOX transcription factor (e.g., about 200, 205, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 311, 313, 314, 315, 316, 317, 318, 319 or 320 consecutive amino acid residues, or any value or range therein). In al Petition 870250103857, dated 11 / 13 / 2025, pp. 98 / 212 89 / 166 In some embodiments, a mutation within the functional WOX gene may comprise a point mutation. In some embodiments, the mutation results in a mutant WOX gene comprising a nucleotide sequence with at least 90% sequence identity with any of the WOX genes mutated with a nucleotide sequence of SEQ ID NOs: 143, 144, 145, 147, 149, 151, 153 and / or 155 and / or results in a mutant WOX gene encoding a WOX transcription factor with at least 90% sequence identity with any of the WOX transcription factors mutated with an amino acid sequence of SEQ ID NOs: 146, 148, 150, 152, 154 and / or 156, optionally, where at least one mutation is an unnatural mutation.

[00207] In some embodiments, the Rosaceae plant cell can be regenerated into a Rosaceae plant comprising at least one mutation, optionally wherein the Rosaceae plant regenerated from the Rosaceae plant cell exhibits a phenotype of a reduced number of spines and / or prickles, spines and / or prickles exhibiting a modified architecture, spines and / or prickles that are reduced in size, and / or absence of spines and / or prickles when compared to a control Rosaceae plant lacking at least one mutation. In some embodiments, a Rosaceae plant comprising at least one mutation in an endogenous functional WOX gene is not regenerated.

[00208] In some embodiments, a method is provided for producing / reproducing an edited, transgene-free Rosaceae plant, comprising the method: crossing a Rosaceae plant of the present invention (for example, a Rosaceae plant comprising one or more mutations (optionally, one or more unnatural mutations) in one or more mutated WOX functional genes and having a reduced number of spines and / or prickles, thorns and / or Petition 870250103857, dated 11 / 13 / 2025, pp. 99 / 212 90 / 166 prickles having modified architecture, spines and / or prickles that are reduced in size and / or without spines and / or prickles (e.g., devoid of spines and / or prickles) with a transgene-free plant, thus introducing the mutation into the Rosaceae plant that is transgene-free; and selecting a Rosaceae parent plant that includes the mutation and is transgene-free, thus producing a transgene-free edited Rosaceae plant.

[00209] Also provided herein is a method for providing a plurality of Rosaceae plants having a reduced number of thorns and / or prickles, thorns and / or prickles having a modified architecture, thorns and / or prickles that are reduced in size and / or no thorns and / or prickles, the method comprising planting two or more Rosaceae plants of the invention (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1000, 2000, 3000, 400, 5000 or 10.000 or more Rosaceae plants comprising one or more mutations (optionally, one or more unnatural mutations) in one or more functional WOX genes and a reduced number of spines and / or prickles, spines and / or prickles having a modified architecture, spines and / or prickles that are reduced in size and / or no spines and / or prickles in a growing area (e.g., a field (e.g., a cultivated field, an agricultural field), a growing chamber, a greenhouse, a recreation area, a lawn and / or a roadside and the like), thus providing a plurality of Rosaceae plants with a reduced number of spines and / or prickles, spines and / or prickles having a modified architecture, spines and / or prickles that are reduced in size and / or no spines and / or prickles compared to a plurality of control Rosaceae plants lacking the mutation.

[00210] In some modalities, a method of creating a Petition 870250103857, dated 11 / 13 / 2025, pp. 100 / 212 91 / 166 mutation in an endogenous Homebox-related WUSCHEL (WUS) (WOX) gene in a Rosaceae plant, comprising: (a) targeting a gene editing system to a portion or region of the WOX gene comprising a sequence with at least 80% sequence identity (e.g., at least approximately 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity) with any of the SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134, optionally where the sequence identity may be at least 85%, or at least 90%, or may be at least 95%, optionally the sequence identity may be 100%;and (b) select a Rosaceae plant comprising a modification located in a gene region having at least 80% sequence identity with any of the following SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134.;

[00211] In some embodiments, a method for generating variation in a functional Homebox-related WUSCHEL (WUS) (WOX) gene is provided, the method comprising: introducing an editing system into a Rosaceae plant cell, wherein the editing system is directed to a region of a functional WOX gene encoding a WOX transcription factor and that comes into contact with the region of the functional WOX gene with the editing system, thereby introducing a mutation in the WOX gene and generating variation in the WOX gene of the Rosaceae plant cell. In some embodiments, the functional WOX gene is a functional WOX1 gene or a functional WOX23 gene. In some embodiments, the functional WOX gene comprises a nucleotide sequence with at least 80% identity. Petition 870250103857, dated 11 / 13 / 2025, pages 101 / 212 92 / 166 sequence (e.g., at least approximately 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity) with any of the SEQ ID NOs: 69, 70, 75, 76, 128, 129, or 162-173, and / or the WOX gene region in which the variation is generated comprises a nucleotide sequence with at least 80% sequence identity with any of the SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125, or 131-135, optionally SEQ ID The sequence numbers (SEQ IDs) 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125, or 134, and / or the WOX transcription factor comprises an amino acid sequence with at least 80% sequence identity with any of the SEQ IDs 71, 77, 130, or 174-182, and / or the region of the WOX transcription factor in which the variation is generated comprises an amino acid sequence with at least 80% sequence identity with any of the SEQ IDs 126, 127, 136, or 137.Optionally, where the sequence identity of the above sequences may be at least 85%, or at least 90%, or may be at least 95%, optionally the sequence identity may be 100%. In some embodiments, the target WOX gene region comprises at least 80% sequence identity with any of the nucleotide sequences of SEQ ID NOs: 78-125 or 131-135, optionally for any of the SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134.

[00212] In some embodiments, contacting the endogenous functional WOX gene region in the Rosaceae plant cell with the editing system produces a Rosaceae plant cell containing an edited endogenous WOX gene in its genome, the method further comprising (a) regenerating a Rosaceae plant from the Rosaceae plant cell; (b) self-fertilizing the Rosaceae plant to Petition 870250103857, dated 11 / 13 / 2025, pp. 102 / 212 93 / 166 produce offspring plants of Rosaceae (E1); (c) analyze offspring Rosaceae plants of (b) for a reduced number of spines and / or prickles, spines and / or prickles that have modified architecture, spines and / or prickles that are reduced in size and / or without spines and / or prickles;(d) select Rosaceae parent plants that have a reduced number of thorns and / or prickles, thorns and / or prickles that have a modified architecture, thorns and / or prickles that are reduced in size and / or no thorns and / or prickles to produce selected Rosaceae parent plants that have a reduced number of thorns and / or prickles, thorns and / or prickles that have a modified architecture, thorns and / or prickles that are reduced in size and / or no thorns and / or prickles compared to a control Rosaceae plant. In some embodiments, the method may further comprise (e) self-pollination of the selected Rosaceae offspring plants from (d) to produce Rosaceae offspring plants (E2); (f) testing of Rosaceae plants descended from (e) for a reduced number of thorns and / or prickles, thorns and / or prickles that have a modified architecture, thorns and / or prickles that are reduced in size and / or absence of thorns and / or prickles;(e) and (g) select Rosaceae parent plants exhibiting a reduced number of spines and / or prickles, spines and / or prickles that have a modified architecture, spines and / or prickles that are reduced in size, and / or no spines and / or prickles to produce selected Rosaceae parent plants exhibiting a reduced number of spines and / or prickles, spines and / or prickles that have a modified architecture, spines and / or prickles that are reduced in size, and / or no spines and / or prickles compared to a control Rosaceae plant, optionally repeating (e) to (g) one or more additional times. In some embodiments, the generation of variation in; Petition 870250103857, dated 11 / 13 / 2025, pp. 103 / 212 The 94 / 166 transcription factor WOX in a Rosaceae plant results in the Rosaceae plant having a reduced number of thorns and / or prickles, thorns and / or prickles that have a modified architecture, thorns and / or prickles that are reduced in size, and / or no thorns and / or prickles at all.

[00213] In some embodiments, a mutation in an endogenous functional WOX gene may result in a WOX nucleic acid mutation with at least 90% sequence identity with any of the mutated WOX genes (e.g., WOX1, WOX23) having a nucleotide sequence of SEQ ID NOs: 143, 144, 145, 147, 149, 151, 153 and / or a mutant WOX polypeptide comprising an amino acid sequence with at least 90% sequence identity with any of the SEQ ID NOs: 146, 148, 150, 152, 154 and / or 156.

[00214] In some embodiments, a method for detecting a mutant WOX gene (a mutation in a functional endogenous WOX gene) in a Rosaceae plant is provided comprising detecting in the Rosaceae plant genome a WOX gene with at least one mutation within a region with at least 80% sequence identity with any of the nucleotide sequences of SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally for any of the SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134, optionally where the sequence identity may be at least 85%, or at least 90%, or may be at least 95%, optionally the sequence identity may be 100%.

[00215] In some embodiments, the detected WOX mutant gene comprises a sequence with at least 90% sequence identity to a WOX mutant nucleic acid with a Petition 870250103857, dated 11 / 13 / 2025, pp. 104 / 212 95 / 166 nucleotide sequence of SEQ ID NOs: 143, 144, 145, 147, 149, 151, 153 and / or 155 and / or encoding a mutated WOX polypeptide comprising an amino acid sequence with at least 90% sequence identity with any of the SEQ ID NOs: 146, 148, 150, 152, 154 and / or 156, optionally where the mutation detected in the WOX gene is an unnatural mutation. In some embodiments, the mutation in the mutant WOX gene generated by the methods of this invention may be located in Exon 1 or Exon 2 of the WOX gene.

[00216] In some embodiments, a method is provided for editing a specific site in the genome of a Rosaceae plant cell, the method comprising: cleaving, in a site-specific manner, a target site within an endogenous Homebox-related WUSCHEL (WUS) gene (WOX) in the Rosaceae plant cell, the endogenous WOX gene: (a) comprising a nucleotide sequence with at least 80% sequence identity (e.g., at least approximately 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity) with any of the SEQ ID NOs: 69, 70, 75, 76, 128, 129 or 162-173; (b) comprising a region with at least 80% sequence identity with a nucleotide sequence of any of the SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally to any of the SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134;(c) encode an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 71, 77, 130, or 174-182; and / or (d) encode a region of an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 126, 127, 136, or 137, optionally where the sequence identity of (a), (b), (c), and / or (d) may be at least 80%; Petition 870250103857, dated 11 / 13 / 2025, pp. 105 / 212 96 / 166 in 85%, or at least 90%, or it can be at least 95%, optionally the sequence identity can be 100%, thus generating an edit in the endogenous WOX gene of the Rosaceae plant cell and producing a Rosaceae plant cell comprising the edit in the endogenous WOX gene. In some embodiments, the endogenous WOX gene is specifically expressed in tissues that give rise to trichomes, thorns and / or prickles, optionally, where the endogenous WOX gene is specifically expressed in vascular tissue, epidermal tissue and / or cells or tissues that give rise to thorns and / or prickles in the Rosaceae plant or part thereof. In some embodiments, the endogenous WOX gene is the endogenous WOX1 gene and / or an endogenous WOX23 gene, optionally where an edit is generated in two or more endogenous WOX genes.

[00217] In some embodiments, an edit in the endogenous functional WOX gene in a Rosaceae plant results in a mutation including, but not limited to, a base deletion, a base substitution, or a base insertion, optionally wherein at least one mutation is an unnatural mutation. In some embodiments, at least one mutation may be located in the first exon or second exon of the functional WOX gene. In some embodiments, the edit may result in at least one mutation that is an insertion of at least one base pair (e.g., 1 base pair for approximately 100 base pairs), optionally wherein the insertion is an out-of-frame insertion.In some modalities, editing can result in at least one mutation that is a deletion, optionally where the deletion is about 1 to about 100 consecutive base pairs in length, for example, about 1 to 50 consecutive base pairs, about 1 to 30 consecutive base pairs, or about 1 to 15 consecutive base pairs in length, optionally about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13. Petition 870250103857, dated 11 / 13 / 2025, pp. 106 / 212 97 / 166 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 consecutive base pairs. A useful deletion or insertion with this invention may be an out-of-frame insertion or an out-of-frame deletion. In some embodiments, an out-of-frame insertion or an out-of-frame deletion may result in a premature stop codon and a truncated protein.In some embodiments, editing a functional WOX gene results in a non-functional WOX gene and / or the WOX transcription factor being truncated, optionally a C-terminal truncation, optionally where the C-terminal truncation is a deletion of about one to about 320 consecutive amino acid residues, optionally about 200-320 consecutive amino acid residues from the C-terminal of the WOX transcription factor (e.g., about 200, 205, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 225, 230, 235, 240, 245, 250, 255, 260,). 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 311, 313, 314, 315, 316, 317, 318, 319, or 320 consecutive amino acid residues, or any value or range therein).

[00218] In some embodiments, a Rosaceae plant comprising editing in its endogenously functional WOX gene exhibits a phenotype of reduced number of spines and / or prickles, spines and / or prickles that have a modified architecture, spines and / or prickles that are reduced in size, and / or no spines and / or prickles. In some embodiments, the editing results in a mutation of the WOX gene comprising a nucleotide sequence with at least 90% sequence identity (e.g., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, optionally the sequence identity may be at least 95%, optionally the sequence identity may be 100%) to any of the mutated WOX genes that possess a nucleotide sequence of SEQ ID Petition 870250103857, dated 11 / 13 / 2025, pp. 107 / 212 98 / 166 SEQ IDs: 143, 144, 145, 147, 149, 151, 153 and / or 155 and / or encoding a polypeptide with at least 90% identity to any of the mutated WOX transcription factors of SEQ IDs: 146, 148, 150, 152, 154 and / or 156.

[00219] In some embodiments, an editing method may further comprise the regeneration of a Rosaceae plant from the Rosaceae plant cell comprising the editing in the endogenous functional WOX gene, thereby producing a Rosaceae plant comprising the editing in its endogenous functional WOX gene (optionally in the first and / or second exon of the functional WOX gene) and having a phenotype of reduced number of spines and / or prickles, spines and / or prickles that have a modified architecture, spines and / or prickles that are reduced in size and / or no spines and / or prickles when compared to a control Rosaceae plant that is devoid of the editing.

[00220] In some embodiments, a method is provided for making a Rosaceae plant, the method comprising (a) contacting a population of Rosaceae plant cells comprising a WUSCHEL (WUS)-related Homebox gene (WOX) with a nuclease linked to a nucleic acid binding domain (e.g., editing system) that binds to a sequence (i) with at least 80% sequence identity (e.g., at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity) to a nucleotide sequence of any one of SEQ ID NOs: 69, 70, 75, 76, 128, 129, or 162-173, (ii) comprising a region with at least 80% identity with any of the SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally for any of the SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134; (iii) encoding a sequence Petition 870250103857, dated 11 / 13 / 2025, pp. 108 / 212 99 / 166 amino acids with at least 80% sequence identity with any of the SEQ ID NOs: 71, 77, 130, or 174-182, and / or (iv) encode a region with at least 80% sequence identity with any of the SEQ ID NOs: 126, 127, 136, or 137, optionally wherein the sequence identity of (i), (ii), (iii), and / or (iv) may be at least 85%, or at least 90%, or may be at least 95%, optionally the sequence identity may be 100%; (b) select a Rosaceae plant cell from the Rosaceae plant cell population in which a functional endogenous WOX gene has been mutated, thereby producing a Rosaceae plant cell comprising a mutation in the endogenous WOX gene; and (c) the growth of the selected Rosaceae plant cell in a Rosaceae plant that has a mutation in the endogenous WOX gene.

[00221] In some embodiments, a method for reducing the number and / or size of thorns and / or prickles, for modifying the architecture of thorns and / or prickles and / or eliminating thorns and / or prickles in a Rosaceae plant is provided, the method comprising (a) contacting a Rosaceae plant cell comprising an endogenous Homebox-related WUSCHEL (WUS) gene (WOX) with a nuclease targeting the endogenous WOX gene, wherein the nuclease is linked to a nucleic acid binding domain (e.g., editing system) that binds to a target site within the endogenous WOX gene, wherein the endogenous WOX gene: (i) comprises a nucleotide sequence with at least 80% sequence identity (e.g., at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity) with any of the SEQ ID NOs: 69, 70, 75, 76, 128, 129 or 162-173;(ii) comprises a region with at least 80% sequence identity with a nucleotide sequence of any of the following SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or; Petition 870250103857, dated 11 / 13 / 2025, pp. 109 / 212 100 / 166 131-135, optionally for any of the SEQ ID Nos: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134; (iii) encodes an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 71, 77, 130, or 174-182; and / or (iv) encodes a region of an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 126, 127, 136 or 137, optionally wherein the sequence identity of (i), (ii), (iii) and / or (iv) may be at least 85%, or at least 90%, or may be at least 95%, optionally the sequence identity may be 100% to produce a Rosaceae plant cell comprising a mutation in the endogenous WOX gene;and (b) the growth of a Rosaceae plant cell comprising a mutation in the endogenous WOX gene in a Rosaceae plant comprising the mutation in the endogenous WOX gene thus producing a Rosaceae plant with an endogenous WOX gene mutation and reduced number of spines and / or prickles, spines and / or prickles with modified architecture, reduced size of spines and / or prickles and / or elimination of spines and / or prickles in the Rosaceae plant.;

[00222] In some embodiments, a method for producing a Rosaceae plant or part thereof comprising at least one cell with an endogenous mutation of the WUSCHEL-related Homebox gene (WUS) (WOX) (e.g., one or more mutated endogenous WOX genes), the method comprising contacting a target site within an endogenous WOX gene in the Rosaceae plant or part thereof with a nuclease comprising a cleavage domain and a nucleic acid-binding domain, wherein the nucleic acid-binding domain binds to a target site within the endogenous WOX gene, wherein the endogenous WOX gene (a) comprises a nucleotide sequence with at least 80% of Petition 870250103857, dated 11 / 13 / 2025, pp. 110 / 212 101 / 166 sequence identity (e.g., at least approximately 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity) with any of the following SEQ ID numbers: 69, 70, 75, 76, 128, 129 or 162-173; (b) comprises a region with at least 80% sequence identity with a nucleotide sequence of any of the following SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally for any of the following SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134; (c) encodes an amino acid sequence with at least 80% sequence identity with any of the following SEQ ID NOs: 71, 77, 130, or 174-182;and / or (d) encodes a region of an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 126, 127, 136 or 137, optionally where the sequence identity of (i), (ii), (iii) and / or (iv) may be at least 85%, or at least 90%, or may be at least 95%, optionally the sequence identity may be 100%, therefore producing the Rosaceae plant or part thereof comprising at least one cell with a mutation in the endogenous WOX gene (e.g., a mutation in one or more endogenous WOX genes).

[00223] Also provided herein is a method for producing a Rosaceae plant or part thereof comprising a mutated endogenous WUSCHEL (WUS)-related Homebox gene (WOX) and exhibiting a reduced number of spines and / or prickles, spines and / or prickles having a modified architecture, spines and / or prickles that are reduced in size and / or no spines and / or prickles, the method comprising contacting a target site within an endogenous WOX gene in the Rosaceae plant or part thereof with a nuclease comprising a cleavage domain and a nucleic acid-binding domain, wherein the nucleic acid-binding domain Petition 870250103857, dated 11 / 13 / 2025, pp. 111 / 212 102 / 166 cleico binds to a target site within the endogenous WOX gene, wherein the endogenous WOX gene: (a) comprises a nucleotide sequence with at least 80% sequence identity (e.g., at least approximately 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity) with any of the SEQ ID NOs: 69, 70, 75, 76, 128, 129 or 162-173; (b) comprises a region with at least 80% sequence identity with a nucleotide sequence of any of the following SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally for any of the following SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134; (c) encodes an amino acid sequence with at least 80% sequence identity with any of the following SEQ ID NOs: 71, 77, 130, or 174-182;and / or (d) encodes a region of an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 126, 127, 136 or 137, optionally where the sequence identity of (i), (ii), (iii) and / or (iv) may be at least 85%, or at least 90%, or may be at least 95%, optionally the sequence identity may be 100%, therefore producing the Rosaceae plant or part thereof comprising an endogenous WOX gene that has a mutation and exhibits a reduced number of spines and / or prickles, spines and / or prickles that exhibit a modified architecture, spines and / or prickles that are reduced in size and / or absence of spines and / or prickles.;

[00224] In some embodiments, a target site may be a region or within a region of the WOX gene (a) having at least 80% sequence identity (e.g., at least approximately 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity, optionally identity Petition 870250103857, dated 11 / 13 / 2025, pp. 112 / 212 (a) having at least 85% sequence identity, or at least 90%, or at least 95%, optionally 100% sequence identity with a nucleotide sequence of any of the following SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125, or 131-135, optionally any of the following SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125, or 134 or (b) having at least less than 80% sequence identity with a sequence of nucleotides encoding any of the amino acid sequences with SEQ IDs: 126, 127, 136 or 137, optionally where the sequence identity of (a) and / or (b) may be at least 85%, or at least 90%, or may be at least 95%, optionally the sequence identity may be 100%.

[00225] In some embodiments, a mutation may be a base substitution, a base deletion, a base insertion, and / or an inversion, optionally an unnatural mutation. In some embodiments, a mutation is a base substitution for an A, a T, a G, or a C. In some embodiments, the mutation provided in the functional WOX gene is a deletion or an insertion, optionally an out-of-frame deletion or an out-of-frame insertion. In some embodiments, a mutation provided in an endogenous functional WOX gene may be a dominant mutation, a semi-dominant mutation, a recessive mutation, or a hypomorphic mutation. In some embodiments, a mutation may be a deletion (e.g., out-of-frame deletion) from about 1 base pair to about 320 consecutive base pairs, optionally 1 to about 50 consecutive base pairs, 1 to about 30 consecutive base pairs, 1 to about 15 consecutive base pairs.In some modalities, a mutation can be an insertion (e.g., an out-of-frame insertion) of at least one ba pair. Petition 870250103857, dated 11 / 13 / 2025, pages 113 / 212 104 / 166 ses (e.g., 1 base pair for approximately 100 consecutive base pairs). In some embodiments, a mutation may be located in the first exon and / or the second exon of the WOX gene. In some embodiments, a mutation provided in a functional WOX gene that is an out-of-frame deletion or an out-of-frame insertion may result in a premature stop codon and a truncated WOX transcription factor. In some embodiments, a mutation provided in an endogenous functional WOX gene may result in the encoded WOX transcription factor having a truncation, optionally a C-terminal truncation. In some embodiments, a mutation provided in an endogenous functional WOX gene may result in a reduced amount of the encoded WOX transcription factor or no detectable encoded WOX transcription factor.

[00226] In some embodiments, a plant or part thereof produced by the methods of this invention comprises an endogenous mutation of the WOX gene as described herein and exhibits a reduced number of spines and / or prickles, spines and / or prickles that have a modified architecture, spines and / or prickles that are reduced in size and / or no spines and / or prickles compared to a control Rosaceae plant that is devoid of the mutation in the endogenous functional WOX gene, for example, the plant or part of the plant has not had a target site within its endogenous functional WOX gene brought into contact with an editing system or a nuclease comprising a cleavage domain and a nucleic acid binding domain (e.g., a DNA binding domain).

[00227] In some embodiments, a mutation in an endogenous functional WOX gene can result in a WOX nucleic acid mutation with at least 90% sequence identity to any of the mutated WOX genes (e.g., WOX1, WOX23) having a nucleotide sequence with SEQ IDs: 143, 144, 145, Petition 870250103857, dated 11 / 13 / 2025, pp. 114 / 212 105 / 166 147, 149, 151, 153 and / or 155 and / or encode a mutated WOX polypeptide comprising an amino acid sequence with at least 90% sequence identity with any of the SEQ ID NOs: 146, 148, 150, 152, 154 and / or 156.

[00228] A nuclease that comes into contact with a plant cell, a population of plant cells, and / or a target site cleaves an endogenous functional WOX gene, thereby introducing a mutation into the endogenous functional WOX gene. A nuclease useful for the invention can be any nuclease that can be used to edit / modify a target nucleic acid. Such nucleases include, without limitation, a zinc finger nuclease, transcription activator-like effector nucleases (TALENs), endonuclease (e.g., Fok1), and / or a CRISPR-Cas effector protein. Similarly, any nucleic acid binding domain useful for the invention can be any DNA binding domain or RNA binding domain that can be used to edit / modify a target nucleic acid.These nucleic acid binding domains include, without limitation, a zinc finger, a transcription activator-like DNA binding domain (TAL), an argonaut, and / or a CRISPR-Cas effector DNA binding domain.

[00229] In some embodiments, a nucleic acid-binding domain (e.g., DNA-binding domain) is contained within a nucleic acid-binding polypeptide. A nucleic acid-binding protein or nucleic acid-binding polypeptide, as used herein, refers to a polypeptide that binds and / or is capable of binding to a nucleic acid in a site-specific and / or sequence-specific manner. In some embodiments, a nucleic acid-binding polypeptide may be a sequence-specific nucleic acid-binding polypeptide (e.g., a sequence-specific DNA-binding domain), such as, without limitation, a Petition 870250103857, dated 11 / 13 / 2025, pp. 115 / 212 106 / 166 polypeptide and / or sequence-specific binding domain, for example, of a polynucleotide-guided endonuclease, a CRISPR-Cas effector protein (e.g., a CRISPRCas endonuclease), a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), and / or an Argonaut protein. In some embodiments, a nucleic acid-binding polypeptide comprises a cleavage polypeptide (e.g., a polypeptide and / or nuclease domain), such as, without limitation, an endonuclease (e.g., Fok1), 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) one or more nucleic acid molecules (e.g., forms a complex with a guide nucleic acid, as described in this document) that can direct 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 one or more nucleic acid molecules (or a portion or region thereof), 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 in this document.In some embodiments, specific reference is made to a CRISPR-Cas effector protein for simplification, but a nucleic acid-binding polypeptide, as described in this document, may be used. In some embodiments, a polynucleotide and / or nucleic acid construct of the invention may be an expression cassette or may be contained within an expression cassette. Petition 870250103857, dated 11 / 13 / 2025, pp. 116 / 212 107 / 166

[00230] In some embodiments, a method for editing an endogenous Homebox-related WUSCHEL (WUS) (WOX) gene (e.g., WOX1 and / or WOX23) in a Rosaceae plant or plant part is provided, the method comprising contacting a target site within an endogenous WOX gene in the Rosaceae plant or plant part with a cytosine base editing system comprising a cytosine deaminase and a nucleic acid binding domain that binds to a target site within the endogenous WOX gene, wherein the endogenous WOX gene: (a) comprises a nucleotide sequence with at least 80% sequence identity (e.g., at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity) with any of the following SEQ ID numbers: 69, 70, 75, 76, 128, 129 or 162-173;(b) comprises a region with at least 80% sequence identity with a nucleotide sequence of any of the following SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally for any of the following SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120; 122, 124, 125, or 134; (c) encodes an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 71, 77, 130, or 174-182; and / or (d) encodes a region of an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 126, 127, 136, or 137, optionally where the sequence identity of (a), (b), (c), and / or (d) may be at least 85%, or at least 90%, or may be at least 95%, optionally the sequence identity may be 100%, thereby editing the endogenous WOX gene in the Rosaceae plant or part thereof and producing a Rosaceae plant or part thereof comprising at least one cell with a mutation in the endogenous WOX gene. In some embodiments, the acid Petition 870250103857, dated 11 / 13 / 2025, pp. 117 / 212 108 / 166 nucleic acid detected may comprise an unnatural mutation.

[00231] In some embodiments, a method for editing an endogenous Homebox-related WUSCHEL (WUS) (WOX) gene (e.g., WOX1 and / or WOX23) in a Rosaceae plant or plant part is provided, the method comprising contacting a target site within a WOX gene in the Rosaceae plant or plant part with an adenosine base editing system comprising an adenosine deaminase and a nucleic acid binding domain that binds to a target site within the WOX gene, wherein the WOX gene (a) comprises a nucleotide sequence with at least 80% sequence identity (e.g., at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity) with any of the following SEQ ID numbers: 69, 70, 75, 76, 128, 129, or 162-173;(b) comprises a region with at least 80% sequence identity with a nucleotide sequence of any of the following SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally for any of the following SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134; (c) encodes an amino acid sequence with at least 80% sequence identity with any of the following SEQ ID NOs: 71, 77, 130, or 174-182;and / or (d) encodes a region of an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 126, 127, 136 or 137, optionally wherein the sequence identity of (a), (b), (c) and / or (d) may be at least 85%, or at least 90%, or may be at least 95%, optionally the sequence identity may be 100%, thereby editing the endogenous WOX gene in the Rosaceae plant or part thereof and producing a Rosaceae plant or part thereof comprising at least one cell with a mutation in the endogenous gene. Petition 870250103857, dated 11 / 13 / 2025, p. 118 / 212; 109 / 166 geno WOX.

[00232] In some embodiments, the present invention provides a method for producing a Rosaceae plant comprising a mutation in an endogenous Homebox-related WUSCHEL (WUS) (WOX) gene (e.g., WOX1 and / or WOX23) and at least one polynucleotide of interest, the method comprising crossing a Rosaceae plant of the invention comprising at least one mutation in an endogenous functional WOX gene (a first Rosaceae plant) with a second Rosaceae plant comprising at least one polynucleotide of interest to produce offspring Rosaceae plants; and selecting offspring Rosaceae plants comprising at least one mutation in the WOX gene and at least one polynucleotide of interest, thereby producing the Rosaceae plant comprising a mutation in an endogenous functional WOX gene and at least one polynucleotide of interest.

[00233] The present invention further provides a method for producing a Rosaceae plant comprising a mutation in an endogenous Homebox gene related to WUSCHEL (WUS) (WOX) (e.g., WOX1 and / or WOX23) and at least one polynucleotide of interest, the method comprising introducing at least one polynucleotide of interest into a Rosaceae plant of the present invention comprising at least one mutation in a functional WOX gene, thereby producing a Rosaceae plant comprising at least one mutation in a WOX gene and at least one polynucleotide of interest.

[00234] In some embodiments, a method is also provided for producing a Rosaceae plant comprising a mutation in an endogenous Homebox gene related to WUSCHEL (WUS) (WOX) (e.g., WOX1 and / or WOX23) and exhibiting a phenotype of Petition 870250103857, dated 11 / 13 / 2025, pp. 119 / 212 110 / 166 a reduced number of thorns and / or prickles, thorns and / or prickles that have a modified architecture, thorns and / or prickles that are reduced in size and / or without thorns and / or prickles, the method comprising crossing a first Rosaceae plant, which is a Rosaceae plant of the present invention comprising at least one mutation in a functional WOX gene, with a second Rosaceae plant that exhibits a phenotype of a reduced number of thorns and / or prickles, thorns and / or prickles that have a modified architecture, thorns and / or prickles that are reduced in size and / or without thorns and / or prickles;and selecting Rosaceae parent plants comprising the mutation in the WOX gene and a phenotype of a reduced number of spines and / or prickles, spines and / or prickles that have a modified architecture, spines and / or prickles that are reduced in size and / or no spines and / or prickles, thus producing the Rosaceae plant comprising a mutation in an endogenous WOX gene and exhibiting a phenotype of a reduced number of spines and / or prickles, spines and / or prickles that have a modified architecture, spines and / or prickles that are reduced in size and / or no spines and / or prickles compared to a control plant.

[00235] In addition, a method is provided for controlling weeds in a container (e.g., pot or seed tray and the like), a growth chamber, a greenhouse, a field, a recreation area, a lawn or a roadside, the method comprising applying a herbicide to one or more (a plurality) Rosaceae plants of the invention (e.g., a Rosaceae plant comprising at least one mutation in a functional WOX gene as described herein) growing in a container, a growth chamber, a greenhouse, a field, a recreation area, a lawn or a roadside, thereby controlling Petition 870250103857, dated 11 / 13 / 2025, pages 120 / 212 111 / 166 the weeds in the container, in the growing chamber, in the greenhouse, in the field, in the recreation area, on the lawn or on the roadside where one or more Rosaceae plants are growing.

[00236] In some embodiments, a method is provided for reducing insect predation on a Rosaceae plant, the method comprising applying an insecticide to one or more Rosaceae plants of the invention, optionally, wherein the one or more Rosaceae plants are growing in a container, a growing chamber, a greenhouse, a field, a recreation area, a lawn or by the roadside, thereby reducing insect predation on the one or more Rosaceae plants.

[00237] In some embodiments, a method is provided for reducing fungal diseases in a Rosaceae plant, the method comprising applying a fungicide to one or more Rosaceae plants of the invention, optionally, wherein the one or more Rosaceae plants are growing in a container, a growing chamber, a greenhouse, a field, a recreation area, a lawn or by the roadside, thereby reducing fungal diseases in the one or more Rosaceae plants.

[00238] A polynucleotide of interest may be any polynucleotide that can confer a desirable phenotype or modify the phenotype or genotype of a plant. In some embodiments, a polynucleotide of interest may be a polynucleotide that confers herbicide tolerance, insect resistance, nematode resistance, disease resistance, increased yield, increased nutrient use efficiency, or resistance to abiotic stress.

[00239] A functional WUSCHEL (WUS) (WOX) related Homebox gene useful with this invention includes any Rosaceae WOX gene in which a mutation such as that described herein can confer a reduced number of spines and / or prickles, a modified architecture Petition 870250103857, dated 11 / 13 / 2025, pp. 121 / 212 112 / 166 [Illegible] ... In some embodiments, a functional endogenous WOX gene (a) comprises a nucleotide sequence with at least 80% sequence identity (e.g., at least approximately 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity) with any of the SEQ ID NOs: 69, 70, 75, 76, 128, 129 or 162-173; (b) comprises a region with at least 80% sequence identity with a nucleotide sequence of any of the following SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally for any of the following SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134; (c) encodes an amino acid sequence with at least 80% sequence identity with any of the following SEQ ID NOs: 71, 77, 130, or 174-182; and / or (d) encodes a region of an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 126, 127, 136 or 137, optionally wherein the sequence identity of (a), (b), (c) and / or (d) may be at least 85%, or at least 90%, or may be at least 95%, optionally the identity of ... Petition 870250103857, dated 11 / 13 / 2025, p. 122 / 212, 113 / 166 of the sequence can be 100%.

[00240] In some embodiments, at least one mutation in an endogenous functional WOX gene in a Rosaceae plant may be a base substitution, a base deletion, a base insertion, and / or an inversion, optionally wherein at least one mutation may be an unnatural mutation. In some embodiments, at least one mutation in an endogenous functional WOX gene in a Rosaceae plant may result in a Rosaceae plant having the phenotype of a reduced number of spines and / or prickles, spines and / or prickles that have a modified architecture, spines and / or prickles that are reduced in size, and / or no spines and / or prickles compared to a control plant devoid of the edit / mutation.

[00241] In some embodiments, a mutation in a functional endogenous WOX gene may be a base substitution, a base deletion, and / or a base insertion of at least 1 base pair. In some forms, a base deletion can range from 1 nucleotide to approximately 100 nucleotides (for example, approximately 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, 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 base pairs, or any range or value therein, for example, 1 to about 50 base pairs, 1 to about 30 base pairs, 1 to about 15 base pairs, or any range or value therein), optionally where the mutation is in about 2 to about 100 consecutive nucleotides (for example, 1 to about 50 consecutive base pairs, 1 to about 30 consecutive base pairs, 1 to about 15 consecutive base pairs). In some modalities Petition 870250103857, dated 11 / 13 / 2025, pp. 123 / 212 114 / 166 des, a mutation in an endogenous functional WOX gene can be a base insertion of 1 to about 100 consecutive nucleotides of the WOX nucleic acid. In some embodiments, a mutation in an endogenous functional WOX gene can be an out-of-frame insertion or an out-of-frame deletion that results in a WOX transcript with a C-terminal truncation. In some embodiments, a mutation in an endogenous functional WOX gene can be an out-of-frame insertion or an out-of-frame deletion that results in a reduced amount of the encoded WOX transcription factor or where the encoded WOX transcription factor is undetectable. In some embodiments, a mutation can be a base substitution, optionally a substitution for an A, a T, a G, or a C. A mutation can be a point mutation. In some embodiments, a mutation can be an unnatural mutation.

[00242] In some embodiments, a mutation in an endogenous WOX gene can be produced after cleavage by an editing system comprising a nuclease and a nucleic acid binding domain that binds to a target site within a functional target nucleic acid (e.g., a functional endogenous WOX gene, e.g., functional WOX1, and / or functional WOX23), the target nucleic acid comprising a sequence with at least 80% sequence identity with any of the nucleotide sequences of SEQ ID NOs: 69, 70, 75, 76, 128, 129, or 162-173, and / or encoding an amino acid sequence with at least 80% sequence identity (e.g., at least approximately 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity) with any of the SEQ ID Nos: 71, 77, 130, or 174-182,optionally where the target site is located in a region of the WOX gene: the region comprising a sequence with at least 80% identity to any of the, Petition 870250103857, dated 11 / 13 / 2025, pp. 124 / 212 115 / 166 SEQ ID Nos: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally SEQ ID Nos: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134, and / or encoding a sequence with at least 80% sequence identity with an amino acid sequence from any of the SEQ ID Nos: 126, 127, 136 or 137, optionally wherein the sequence identity of the target nucleic acid, region and / or encoded polypeptide may be at least 85%, or at least 90%, or it could be at least 95%, optionally the sequence identity could be 100%.

[00243] In addition, guide nucleic acids (e.g., gRNA, gDNA, crRNA, crDNA) are provided that bind to a target site within a WUSCHEL-related endogenous Homebox gene (WUS) (WOX) (e.g., WOX1 and / or WOX23), the endogenous gene (a) comprising a sequence with at least 80% sequence identity (e.g., at least approximately 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity) with any of the nucleotide sequences of SEQ ID NOs: 69, 70, 75, 76, 128, 129 or 162-173; (b) comprising a region with at least 80% sequence identity with a nucleotide sequence of any of the SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134;(c) encode an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 71, 77, 130, or 174-182; and / or (d) encode a region of an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 126, 127, 136, or 137, optionally where the sequence identity of (a), (b), (c), and / or (d) may be at least; Petition 870250103857, dated 11 / 13 / 2025, pp. 125 / 212 116 / 166 85%, or at least 90%, or it could be at least 95%, optionally the sequence identity could be 100%. In some embodiments, the target site is within a region of the WOX gene with at least 80% sequence identity with any of the following SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125, or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125, or 134 (for example, it may comprise a consecutive nucleotide portion of any one or more nucleotide sequences from any of the following SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134, or it may comprise a portion of consecutive nucleotides of a nucleic acid encoding the amino acid sequence of any of the SEQ ID NOs: 126, 127, 136 or 137),Optionally, where sequence identity may be at least 85%, or at least 90%, or at least 95%, optionally sequence identity may be 100%. In some embodiments, a guide nucleic acid may comprise a spacer comprising any of the nucleotide sequences with SEQ ID numbers: 138-142.

[00244] Useful spacer sequences with a guide of this invention comprise complementarity to a fragment or portion of a nucleotide sequence with at least 80% sequence identity (for example, at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity) to any of the nucleotide sequences with SEQ ID NOs: 69, 70, 75, 76, 128, 129, or 162-173 and / or SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally SEQ ID Nos: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134, optionally in Petition 870250103857, dated 11 / 13 / 2025, pp. 126 / 212 117 / 166 wherein the sequence identity may be at least 85%, or at least 90%, or may be at least 95%, optionally the sequence identity may be 100%; or a fragment or portion of a nucleotide sequence encoding a polypeptide comprising a sequence with at least 80% sequence identity with the amino acid sequence of any of SEQ ID NOs: 71, 77, 130, or 174-182 and / or SEQ ID NOs: 126, 127, 136 or 137, optionally wherein the sequence identity may be at least 85%, or at least 90%, or may be at least 95%, optionally the sequence identity may be 100%.In some embodiments, a guide nucleic acid may comprise a spacer with a nucleotide sequence having at least 80% sequence identity with any of the SEQ ID NOs: 138142, or any combination thereof, optionally wherein the sequence identity may be at least 85%, or at least 90%, or may be at least 95%, optionally the sequence identity may be 100%.

[00245] In some embodiments, a Rosaceae plant or part of a plant comprising at least one mutation in at least one functional endogenous Homebox-related WUSCHEL (WUS) gene (WOX) (e.g., WOX1 and / or WOX23) is provided. In some embodiments, at least one mutation may be an unnatural mutation. In some embodiments, the mutation results in a mutant WOX gene comprising a sequence with at least 90% identity to any of the SEQ ID NOs: 143, 144, 145, 147, 149, 151, 153 and / or 155, optionally wherein the percentage of identity to SEQ ID NOs: 143, 144, 145, 147, 149, 151, 153 and / or 155 may be at least 95% or may be 100%.

[00246] In some embodiments, a system is provided comprising a guide nucleic acid that binds to a target site within Petition 870250103857, dated 11 / 13 / 2025, pp. 127 / 212 118 / 166 of a WOX gene, where the target site is in a region of the WOX gene with at least 80% sequence identity (e.g., at least approximately 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity) with any of the SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134, optionally wherein the sequence identity may be at least 85%, or at least 90%, or may be at least 95%, optionally the sequence identity may be 100%, and / or comprising a spacer (e.g., one or more spacers) having the nucleotide sequence of any of the SEQ ID NOs: 138142, and a CRISPR-Cas effector protein that associates with the guide nucleic acid.In some embodiments, the system may also comprise a tracr nucleic acid that associates with the guide nucleic acid and a CRISPR-Cas effector protein, optionally in which the tracr nucleic acid and the guide nucleic acid are covalently linked.

[00247] As used herein, a CRISPR-Cas effector protein in association with a guide nucleic acid refers to the complex that is formed between a CRISPR-Cas effector protein and a guide nucleic acid to direct the CRISPR-Cas effector protein to a target site within a gene. The invention further provides a gene-editing system comprising a CRISPR-Cas effector protein in association with a guide nucleic acid, and the guide nucleic acid comprises a spacer sequence that binds to a functional WUSCHEL-related Homebox (WUS) gene (WOX), optionally wherein the WOX gene: (a) comprises a nucleotide sequence with at least 80% sequence identity (e.g., at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity) with which Petition 870250103857, dated 11 / 13 / 2025, pp. 128 / 212 119 / 166 requires one of the following SEQ ID NOs: 69, 70, 75, 76, 128, 129, or 162-173; (b) comprises a region with at least 80% sequence identity with a nucleotide sequence from any of the following SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125, or 131-135, optionally for any of the following SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125, or 134; (c) encodes an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 71, 77, 130, or 174-182; and / or (d) encodes a region of an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 126, 127, 136, or 137, optionally wherein the sequence identity of (a), (b), (c), and / or (d) may be at least 85%, or at least 90%, or may be at least 95%, optionally the sequence identity may be 100%.In some embodiments, a guide nucleic acid spacer sequence may comprise the nucleotide sequence of any of the SEQ ID NOs: 138-142. In some embodiments, the gene editing system may further include a tracr nucleic acid that associates with the guide nucleic acid and a CRISPR-Cas effector protein, optionally in which the tracr nucleic acid and the guide nucleic acid are covalently linked.

[00248] The present invention further provides a complex comprising a CRISPR-Cas effector protein comprising a cleavage domain and a guide nucleic acid, wherein the guide nucleic acid binds to a target site within an endogenous WUSCHEL-related Homebox gene (WOX), wherein the endogenous WOX gene: (a) comprises a nucleotide sequence with at least 80% sequence identity (for example, at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity) with any of the SEQs Petition 870250103857, dated 11 / 13 / 2025, pp. 129 / 212 120 / 166 (a) comprises a nucleotide sequence with at least 80% sequence identity with any of the following nucleotide sequences: 69, 70, 75, 76, 128, 129 or 162-173; (b) comprises a region with at least 80% sequence identity with any of the following nucleotide sequences: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally with any of the following nucleotide sequences: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134; (c) encodes an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 71, 77, 130, or 174-182; and / or (d) encodes a region of an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 126, 127, 136, or 137, optionally wherein the sequence identity of (a), (b), (c), and / or (d) may be at least 85%, or at least 90%, or may be at least 95%, optionally the sequence identity may be 100%, and the cleavage domain cleaves a target strand in the WOX gene.

[00249] In some embodiments, an expression cassette is provided comprising (a) a polynucleotide encoding the CRISPR-Cas effector protein comprising a cleavage domain and (b) a guide nucleic acid that binds to a target site within an endogenous Homebox-related WUSCHEL (WUS) (WOX) gene in Rosaceae, wherein the guide nucleic acid comprises a spacer sequence that is complementary to and binds to (i) a portion of a nucleic acid with at least 80% sequence identity (e.g., at least approximately 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity) with any of the SEQ ID NOs: 69, 70, 75, 76, 128, 129 or 162-173;(ii) a portion of a nucleic acid with at least 80% sequence identity with any of the SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally for any of the SEQ ID NOs: 81, 82, 84-86, 88-90, 94 Petition 870250103857, dated 11 / 13 / 2025, p. 130 / 212; 121 / 166 97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134; (iii) a portion of a nucleic acid that encodes an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 71, 77, 130, or 174-182; and / or (iv) a portion of a nucleic acid that encodes an amino acid sequence with at least 80% identity to any of the SEQ ID NOs: 126, 127, 136 or 137, optionally wherein the sequence identity of (i), (ii), (iii) and / or (iv) may be at least 85%, or at least 90%, or may be at least 95%, optionally the sequence identity may be 100%.

[00250] Nucleic acids encoding a homeobox (WOX) transcription factor related to WUSCHEL (WUS) (e.g., WOX1, WOX23) are also provided, optionally wherein, when present in a Rosaceae plant or plant part, the mutant WOX gene results in the Rosaceae plant comprising a phenotype of reduced number of spines and / or prickles, spines and / or prickles that have a modified architecture, spines and / or prickles that are reduced in size and / or no spines and / or prickles compared to a control Rosaceae plant or plant part devoid of the mutation.In some embodiments, a WOX mutant gene has at least 90% sequence identity (e.g., at least approximately 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity; optionally, the sequence identity may be at least 95%; optionally, the sequence identity may be 100%) to any of the WOX mutant genes (e.g., WOX1, WOX23) with SEQ ID NOs: 143, 144, 145, 147, 149, 151, 153 and / or 155 and / or encodes a mutated WOX polypeptide comprising an amino acid sequence with at least 90% sequence identity to any of the SEQ ID NOs: 146, 148, 150, 152, 154 and / or 156. Petition 870250103857, dated 11 / 13 / 2025, pp. 131 / 212 122 / 166

[00251] Genetic markers are also provided for use on a blackberry plant or part thereof to identify plants with thorns and / or prickles with a modified architecture, a reduced number and / or size of thorns and / or prickles, or without thorns and / or prickles, the genetic marker comprising (a) the nucleic acid sequence GAACTCTG located between nucleotide positions 2897 and 2898 with reference to the nucleotide position numbering of SEQ ID NO: 69 and / or (b) the nucleic acid sequence of SEQ ID NO: 143. In some embodiments, the blackberry plant may be Rubus allegheniensis (Allegheny blackberry), Rubus armeniacus (Himalayan blackberry), Rubus canadensis (Canadian blackberry), Rubus fruticosus agg. (European mulberry), Rubus laciniatus (perennial cut-leaf blackberry), Rubus pensilvanicus (Pennsylvanian blackberry), Rubus trifidus (Japanese mulberry), Rubus ursinus (wild mulberry).In some embodiments, a genetic marker, as described herein, can be used to identify a cell of a blackberry plant or part thereof that carries the genetic marker and is therefore capable of being regenerated into a plant with the phenotype of having thorns and / or prickles with a modified architecture, a reduced number and / or size of thorns and / or prickles, or no thorns and / or prickles. In some embodiments, plants or plant parts comprising the genetic markers of the invention are provided.

[00252] In some embodiments, a method is provided for identifying a blackberry plant or part thereof having a thorn and / or prickle phenotype with a modified architecture, a reduced number and / or size of thorns and / or prickles, or no thorns and / or prickles, the method comprising identifying in the genome of a blackberry plant cell or part thereof (a) an insertion in an endogenous Homebox gene related to Petition 870250103857, dated 11 / 13 / 2025, pages 132 / 212 123 / 166 WUSCHEL (WUS) (WOX), the insert comprising the nucleic acid sequence of GAACTCTG located between nucleotide positions 2897 and 2898 with reference to the nucleotide position numbering of SEQ ID NO: 69 and / or (b) the nucleic acid sequence of SEQ ID NO: 143. In some embodiments, plants or plant parts are provided that are identified using the genetic markers of the invention.

[00253] In some embodiments, the present invention further provides a method for cultivating a blackberry plant with thorns and / or prickles with modified architecture, reduced number and / or size of thorns and / or prickles, or without thorns and / or prickles, the method comprising (a) crossing a blackberry plant containing in its genome (i) an insertion in an endogenous Homebox gene related to WUSCHEL (WUS) (WOX), the insertion comprising the nucleic acid sequence of GAACTCTG located between nucleotide positions 2897 and 2898 with reference to the nucleotide position numbering of SEQ ID NO: 69 and / or (ii) the nucleic acid sequence of SEQ ID NO: 143 with itself or with another blackberry plant to produce parent plants;and (b) selecting one or more parent plants comprising the insertion and having thorns and / or prickles with a modified architecture, a reduced number and / or size of thorns and / or prickles, or no thorns and / or prickles, thus producing blackberry plants having thorns and / or prickles with a modified architecture, a reduced number and / or size of thorns and / or prickles, or no thorns and / or prickles. In some embodiments, a method for producing a blackberry plant with thorns and / or prickles with a modified architecture, a reduced number and / or size of thorns and / or prickles, or no thorns and / or prickles may further comprise (c) crossing a selected progeny plant from (b) with itself or; Petition 870250103857, dated 11 / 13 / 2025, pages 133 / 212 124 / 166 with another blackberry plant one or more additional times and select progeny plants comprising the insertion and having thorns and / or prickles with a modified architecture, a reduced number and / or size of thorns and / or prickles, or no thorns and / or prickles, thus producing blackberry plants with thorns and / or prickles with a modified architecture, having a reduced number and / or size of thorns and / or prickles, or having no thorns and / or prickles. In some embodiments, the blackberry plant is Rubus allegheniensis (Allegheny blackberry), Rubus armeniacus (Himalayan blackberry), Rubus canadensis (Canadian blackberry), Rubus fruticosus agg. (European mulberry), Rubus laciniatus (perennial cut-leaf blackberry), Rubus pensilvanicus (Pennsylvanian blackberry), Rubus trifidus (Japanese mulberry), Rubus ursinus (wild mulberry).

[00254] In some embodiments, a blackberry plant or part thereof is provided produced by the identification and improvement methods as described herein and exhibiting a thorn and / or prickle phenotype with a modified architecture, a reduced number and / or size of thorns and / or prickles, or no thorns and / or prickles compared to a control blackberry plant lacking the insertion.

[00255] Nucleic acid constructs of the invention (for example, a construct comprising a sequence-specific nucleic acid binding domain (e.g., sequence-specific DNA binding domain), a CRISPR-Cas effector domain, a deaminase domain, reverse transcriptase (RT), RT template and / or a guide nucleic acid, etc.) and expression cassettes / vectors comprising the same can be used as an editing system of this invention to modify functional target nucleic acids (e.g., functional endogenous WOX genes, e.g., functional endogenous gene WOX1, functional endogenous gene WOX23) and / or their Petition 870250103857, dated 11 / 13 / 2025, pp. 134 / 212 125 / 166 expression.

[00256] Any Rosaceae plant containing an endogenous WOX gene that is capable of conferring a reduced number of spines and / or prickles, spines and / or prickles that have a modified architecture, spines and / or prickles that are reduced in size and / or no spines and / or prickles when a functional WOX gene is modified as described herein, can be modified (e.g., mutated, e.g., edited at the base, cleaved, cut, etc.) as described herein (e.g., using the polypeptides, polynucleotides, RNPs, nucleic acid constructs, expression cassettes and / or vectors of the invention) to modify the architecture of spines and / or prickles, to reduce the number and size of spines and / or prickles and / or to eliminate spines and / or prickles in the Rosaceae plant.

[00257] A useful editing system with this invention may be any currently known or subsequently developed site-specific (sequence-specific) genome editing system that can introduce mutations in a target-specific manner. For example, an editing system (e.g., site-specific or sequence-specific editing system) may 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 primer editing system, each of which may comprise one or more polypeptides and / or one or more polynucleotides that, when expressed as a system in a cell, can modify (change) a target nucleic acid in a sequence-specific manner.In some modes, an editing system (e.g., site- or sequence-specific editing system) may comprise... Petition 870250103857, dated 11 / 13 / 2025, pages 135 / 212 126 / 166 of one or more polynucleotides and / or one or more polypeptides, including, among others, a nucleic acid binding domain (DNA binding domain), a nuclease and / or another polypeptide and / or a polynucleotide.

[00258] In some embodiments, an editing system may comprise one or more sequence-specific nucleic acid binding domains (DNA binding domains) which may be, for example, a polynucleotide-guided endonuclease, a CRISPR-Cas endonuclease (e.g., CRISPRCas effector protein), a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), and / or an Argonaute protein. In some embodiments, an editing system may comprise one or more cleavage domains (e.g., nucleases), including, among others, an endonuclease (e.g., Fok1), 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).In some embodiments, an editing system may comprise one or more polypeptides that include, among others, a deaminase (e.g., a cytosine deaminase, an adenine deaminase), a reverse transcriptase, a Dna2 polypeptide, and / or a 5' flap endonuclease (FEN). In some embodiments, an editing system may comprise one or more polynucleotides, including, among others, a CRISPR matrix nucleic acid (CRISPR guide), an extended guide nucleic acid, and / or a reverse transcriptase template.

[00259] In some embodiments, a method of modifying or editing a functional Homebox gene related to WUSCHEL (WUS) (WOX) may comprise contact with a target nucleic acid (e.g., a nucleic acid encoding the transcription factor Petition 870250103857, dated 11 / 13 / 2025, pp. 136 / 212 127 / 166 WOX, for example, a WOX1 polypeptide, a WOX23 polypeptide) with a base-editing fusion protein (e.g., a sequence-specific DNA-binding protein (e.g., a CRISPR-Cas effector protein or domain) fused to a deaminase domain (e.g., an adenine deaminase and / or a cytosine deaminase) and a guide nucleic acid, wherein the guide nucleic acid is able to guide / direct the base-editing fusion protein to the target nucleic acid, thereby editing a locus within the target nucleic acid. In some embodiments, a base-editing fusion protein and a guide nucleic acid may be comprised in one or more expression cassettes. In some embodiments, the target nucleic acid may be contacted with a base-editing fusion protein and an expression cassette comprising a guide nucleic acid.In some embodiments, sequence-specific nucleic acid-binding fusion proteins and guides can be provided as ribonucleoproteins (RNPs). In some embodiments, a cell can be brought into contact with more than one base-editing fusion protein and / or one or more guide nucleic acids that can target one or more target nucleic acids in the cell.

[00260] In some embodiments, a method of modifying or editing a functional WUSCHEL-related Homebox (WUS) gene (WOX) may comprise contacting a target nucleic acid (e.g., a nucleic acid encoding a WOX polypeptide) with a sequence-specific nucleic acid-binding fusion protein (e.g., a sequence-specific DNA-binding protein (e.g., a CRISPRCas effector protein or domain) fused to a peptide tag, a deaminase fusion protein comprising a deaminase domain (e.g., an adenine deaminase and / or a cytosine deaminase) fused to an affinity polypeptide that is capable of binding to the peptide tag. Petition 870250103857, dated 11 / 13 / 2025, pages 137 / 212 128 / 166 tip and a guide nucleic acid, wherein the guide nucleic acid is able to guide / direct the sequence-specific nucleic acid-binding fusion protein to the target nucleic acid, and the sequence-specific nucleic acid-binding fusion protein is able to recruit the deaminase fusion protein to the target nucleic acid through peptide tag-affinity polypeptide interaction, thus editing a locus within the target nucleic acid. In some embodiments, the sequence-specific nucleic acid-binding fusion protein can be fused to the peptide tag, and the affinity polypeptide that binds to the peptide tag and deaminase can be fused to the peptide tag, thus recruiting the deaminase to the sequence-specific nucleic acid-binding fusion protein and to the target nucleic acid.In some embodiments, the sequence-specific nucleic acid-binding fusion protein, the deaminase fusion protein, and the guide nucleic acid may be contained in one or more expression cassettes. In some embodiments, the target nucleic acid may be brought into contact with a sequence-specific nucleic acid-binding fusion protein, a deaminase fusion protein, and an expression cassette containing a guide nucleic acid. In some embodiments, sequence-specific nucleic acid-binding fusion proteins, deaminase fusion proteins, and guides may be delivered as ribonucleoproteins (RNPs).

[00261] In some embodiments, methods such as primary editing can be used to generate a mutation in a functional endogenous WOX gene in a Rosaceae plant or part thereof. In primary editing, RNA-dependent DNA polymerase (reverse transcriptase, RT) and reverse transcriptase templates (RT template) are used in combination with sequence-specific nucleic acid binding domains that confer the ability to recognize and bind the Petition 870250103857, dated 11 / 13 / 2025, pages 138 / 212 129 / 166 target in a sequence-specific manner, and which may also cause a cut in the strand containing PAM within the target. The nucleic acid binding domain may be a CRISPR-Cas effector protein and, in this case, the CRISPR or guide RNA assembly may be an extended guide comprising an extended portion comprising a primer binding site (PSB) and the edit to be incorporated into the genome (the template). Similar to base editing, core editing may leverage the vanmarkers of various protein recruitment methods for use in target-site editing, such methods including covalent and non-covalent interactions between proteins and nucleic acids used in the selected genome editing process.

[00262] As used herein, a CRISPR-Cas effector protein is a protein or polypeptide or domain thereof that cleaves or cuts a nucleic acid, binds a nucleic acid (e.g., a target nucleic acid and / or a guide nucleic acid) and / or 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.) or part thereof and / or may function as an enzyme.In some embodiments, a CRISPR-Cas effector protein refers to a CRISPR-Cas nuclease polypeptide or domain thereof comprising nuclease activity or in which nuclease activity has been reduced or eliminated and / or comprising nickase activity or in which nickase activity has been reduced or eliminated and / or comprising single-stranded DNA cleavage activity (ss DNase activity) or in which ss DNase activity has been reduced or eliminated and / or comprising self-processing RNase activity or in which self-processing RNase activity has been reduced or eliminated. A CRISPR-Cas effector protein may bind to a target nucleic acid.

[00263] In some embodiments, an acid-binding domain Petition 870250103857, dated 11 / 13 / 2025, pp. 139 / 212 130 / 166 specific sequence nucleic acid can be a CRISPR-Cas effector protein. In some embodiments, a CRISPR-Cas effector protein can be of a Type I CRISPR-Cas system, a Type II CRISPR-Cas system, a Type III CRISPR-Cas system, a Type IV CRISPR-Cas system, a Type V CRISPR-Cas system, or a Type VI CRISPR-Cas system. In some embodiments, a CRISPR-Cas effector protein of the invention can be of a Type II CRISPR-Cas system or a Type V CRISPR-Cas system. In some embodiments, a CRISPR-Cas effector protein can be a Type II CRISPR-Cas effector protein, for example, a Cas9 effector protein. In some embodiments, a CRISPR-Cas effector protein can be a Type V CRISPR-Cas effector protein, for example, a Cas12 effector protein.

[00264] In some embodiments, a CRISPRCas effector protein may include, but is not limited to, Cas9, C2c1, C2c3, Cas12a (also known as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Cas1, Cas1B, Cas2, Cas3, Cas3', Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Nuclease Csf4 (dinG) and / or Csf5, optionally wherein the CRISPR-Cas effector protein may be a Cas9, Cas12a (Cpf1), Cas12b, Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12g, Cas12h, Cas12i, C2c4, C2c5, C2c8, C2c9, C2c10, Cas14a, Cas14b and / or Cas14c effector protein.

[00265] In some embodiments, a useful CRISPRCas effector protein with the invention may comprise a mutation in its nuclease active site (e.g., RuvC, HNH, e.g., RuvC site of a Cas12a nuclease domain; e.g., RuvC site and / or site Petition 870250103857, dated 11 / 13 / 2025, pages 140 / 212 131 / 166 HNH of a Cas9 nuclease domain). A CRISPR-Cas effector protein that has a mutation in its nuclease active site and therefore no longer comprises nuclease activity is commonly called dead, for example, dCas. In some embodiments, a CRISPR-Cas effector protein domain or polypeptide with a mutation in its nuclease active site may have impaired or reduced activity compared to the same CRISPR-Cas effector protein without the mutation, for example, a nickase, e.g., Cas9 nickase, Cas12a nickase.

[00266] A CRISPR Cas9 effector protein or CRISPR Cas9 effector domain useful with this invention may be any known or subsequently identified Cas9 nuclease. In some embodiments, a CRISPR Cas9 polypeptide may be a Cas9 polypeptide from, for example, Streptococcus spp. (e.g., S. pyogenes, S. thermophilus), Lactobacillus spp., Bifidobacterium spp., Kandleria spp., Leuconostoc spp., Oenococcus spp., Pediococcus spp., Weissella spp., and / or Olsenella spp. Example Cas9 sequences include, among others, the amino acid sequences of SEQ ID NO:56 and SEQ ID NO:57 or the nucleotide sequences of SEQ ID NOs: 58-68.

[00267] In some embodiments, the CRISPR-Cas effector protein may be a Cas9 polypeptide derived from Streptococcus pyogenes and recognizes the PAM NGG, NAG, NGA sequence motif (Mali et al, Science 2013; 339(6121): 823-826). In some embodiments, the CRISPR-Cas effector protein may be a Cas9 polypeptide derived from Streptococcus thermophiles and recognizes the PAM NGGNG and / or NNAGAAW (W = A or T) sequence motif (See, for example, Horvath et al, Science, 2010; 327(5962): 167-170, and Deveau et al, J Bacteriol 2008; 190(4): 1390-1400). In some embodiments, the CRISPR-Cas effector protein may be a Cas9 polypeptide derived from Streptococcus mutans and recognizes the PAM NGG sequence motif. Petition 870250103857, dated 11 / 13 / 2025, pp. 141 / 212 132 / 166 and / or NAAR (R = A or G) (See, for example, Deveau et al, J BACTERIOL 2008; 190(4): 1390-1400). In some embodiments, the CRISPR-Cas effector protein may be a Cas9 polypeptide derived from Streptococcus aureus and recognizes the PAM NNGRR sequence motif (R = A or G). In some embodiments, the CRISPR-Cas effector protein may be a Cas9 protein derived from S. aureus, which recognizes the PAM NGRRT sequence motif (R = A or G). In some embodiments, the CRISPR-Cas effector protein may be a Cas9 polypeptide derived from S. aureus, which recognizes the PAM NGRRV sequence motif (R = A or G). In some embodiments, the CRISPR-Cas effector protein may be a Cas9 polypeptide derived from Neisseria meningitidis and recognizes the PAM NGATT or NGCTT sequence motif (R = A or G, V = A, G or C) (See, for example, Hou et al., PNAS 2013, 1-6). In the embodiments mentioned above, N may be any nucleotide residue, for example, any one of A, G, C or T.In some embodiments, the CRISPR-Cas effector protein may be a Cas13a protein derived from Leptotrichia shahii, which recognizes a protospacer flanking sequence (PFS) motif (or RNA PAM (rPAM)) of a single A, U, or C 3', which may be located within the target nucleic acid.

[00268] In some embodiments, the CRISPR-Cas effector protein may be derived from Cas12a, which is a CRISPR-Cas nuclease of regularly spaced short palindromic repeats in V-type clusters, see, for example, amino acid sequences of SEQ ID NOs: 1-17, nucleic acid sequences of SEQ ID NOs: 18-20. Cas12a differs in several respects from the better-known CRISPR nuclease Cas9 Type II. For example, Cas9 recognizes a G-rich protospacer (PAM) adjacent motif that is 3' relative to the binding site of its guide RNA (gRNA, sgRNA, crRNA, crDNA, CRISPR array) (protospacer, target nucleic acid, target DNA) (3'-NGG). Petition 870250103857, dated 11 / 13 / 2025, pages 142 / 212 133 / 166 while Cas12a recognizes a T-rich PAM that is located 5' relative to the target nucleic acid (5'-TTN, 5'-TTTN). In fact, the orientations in which Cas9 and Cas12a bind their guide RNAs are almost reversed with respect to their N and C termini. Furthermore, Cas12a enzymes use a single guide RNA (gRNA, CRISPR array, crRNA) instead of the double guide RNA (sgRNA (e.g., crRNA and tracrRNA)) found in natural Cas9 systems, and Cas12a processes its own gRNAs. Additionally, Cas12a nuclease activity produces staggered breaks in the DNA double strand instead of the blunt ends produced by Cas9 nuclease activity, and Cas12a relies on a single RuvC domain to cleave both DNA strands, while Cas9 utilizes an HNH domain and a RuvC domain for cleavage.

[00269] A useful CRISPR Cas12a effector protein / domain with this invention may be any known or subsequently identified Cas12a polypeptide (formerly known as Cpf1) (see, for example, U.S. Patent No. 9,790,490, which is incorporated by reference for its Cpf1 (Cas12a) sequence disclosures). The term Cas12a, Cas12a polypeptide, or Cas12a domain refers to an RNA-guided nuclease comprising a Cas12a polypeptide, or a fragment thereof, comprising the Cas12a guide nucleic acid binding domain and / or an active, inactive, or partially active Cas12a DNA cleavage domain. In some embodiments, a useful Cas12a with the invention may comprise a mutation in the nuclease active site (e.g., the RuvC site of the Cas12a domain). A Cas12a domain or Cas12a polypeptide that has a mutation in its nuclease active site and therefore no longer comprises nuclease activity is commonly called deadCas12a (e.g., dCas12a).In some forms, a Cas12a domain or Cas12a polypeptide with a mutation at its site. Petition 870250103857, dated 11 / 13 / 2025, pages 143 / 212 134 / 166 active nuclease may have impaired activity, for example, it may have nickase activity.

[00270] Any deaminase domain / polypeptide useful for base editing can be used with this invention. In some embodiments, the deaminase domain may be a cytosine deaminase domain or an adenine deaminase domain. A cytosine deaminase (or cytidine deaminase) useful with this invention may be any known or subsequently identified cytosine deaminase from any organism (see, for example, U.S. Patent No. 10,167,457 and Thuronyi et al. Nat. Biotechnol. 37:1070-1079 (2019), each of which is incorporated by reference herein for its disclosure of cytosine deaminases). Cytosine deaminases can catalyze the hydrolytic deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. Thus, in some embodiments, a useful deaminase or deaminase domain with this invention may be a cytidine deaminase domain, catalyzing the hydrolytic deamination of cytosine to uracil.In some embodiments, a cytosine deaminase may be a variant of a natural cytosine deaminase, including, but not limited to, a primate (e.g., a human, monkey, chimpanzee, gorilla), a dog, a cow, a rat, or a mouse. Thus, in some embodiments, a useful cytosine deaminase with the invention may be about 70% to about 100% identical to a wild-type cytosine deaminase (for example, 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 100% identical, and any range or value therein, to a naturally occurring cytosine deaminase).

[00271] In some embodiments, a useful cytosine deaminase with the invention may be a deaminase family of the complex of Petition 870250103857, dated 11 / 13 / 2025, pp. 144 / 212 135 / 166 apolipoprotein B mRNA editing (APOBEC). In some embodiments, the cytosine deaminase may be an APOBEC1 deaminase, an APOBEC2 deaminase, an APOBEC3A deaminase, an APOBEC3B deaminase, an APOBEC3C deaminase, an APOBEC3D deaminase, an APOBEC3F deaminase, an APOBEC3G deaminase, an APOBEC3H deaminase, an APOBEC4 deaminase, a human activation-induced deaminase (hAID), a rAPOBEC1, FERNY and / or a CDA1, optionally a pmCDA1, an atCDA1 (e.g., At2g19570) and evolved versions thereof (e.g., SEQ ID NO: 27, SEQ ID NO:28 or SEQ ID NO:29). In some embodiments, the cytosine deaminase may be an APOBEC1 deaminase with the amino acid sequence SEQ ID NO:23. In some embodiments, the cytosine deaminase may be an APOBEC3A deaminase with the amino acid sequence SEQ ID NO:24. In some embodiments, the cytosine deaminase may be a CDA1 deaminase, optionally a CDA1 with the amino acid sequence SEQ ID NO:25.In some embodiments, the cytosine deaminase may be a FERNY deaminase, optionally a FERNY with the amino acid sequence SEQ ID NO:26. In some embodiments, a useful cytosine deaminase with the invention may be about 70% to about 100% identical (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%, 99.5% or 100% identical) to the amino acid sequence of a natural cytosine deaminase (e.g., an evolved deaminase). In some embodiments, a useful cytosine deaminase with the invention may be about 70% to about 99.5% identical (for example, 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 99.5%). Petition 870250103857, dated 11 / 13 / 2025, pp. 145 / 212 136 / 166 identical) to the amino acid sequence of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:26 (for example, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to the amino acid sequence of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29). In some embodiments, a polynucleotide encoding a cytosine deaminase can be codon-optimized for expression in a plant, and the codon-optimized polypeptide can be about 70% to 99.5% identical to the reference polynucleotide.

[00272] In some embodiments, a nucleic acid construct of this invention may further encode a uracil glycosylase (UGI) inhibitor polypeptide / domain (e.g., uracil-DNA glycosylase inhibitor). Thus, in some embodiments, a nucleic acid construct encoding a CRISPR-Cas effector protein and a cytosine deaminase domain (e.g., encoding a fusion protein comprising a CRISPR-Cas effector protein domain fused to a cytosine deaminase domain and / or a CRISPR-Cas effector protein domain fused to a peptide marker or an affinity polypeptide capable of binding to a peptide marker and / or a deaminase protein domain fused to a peptide marker or an affinity polypeptide capable of binding to a peptide marker) may further encode a uracil-DNA glycosylase inhibitor (UGI), optionally wherein the UGI may have a codon optimized for expression in a plant.In some embodiments, the invention provides fusion proteins comprising a CRISPR-Cas effector polypeptide, a deaminase domain and a UGI and / or one or more polynucleotides encoding the same, optionally wherein the one or more polynucleotides may have codon optimized for e.g. Petition 870250103857, dated 11 / 13 / 2025, pages 146 / 212 137 / 166 pressure in a plant. In some embodiments, the invention provides fusion proteins wherein a CRISPR-Cas effector polypeptide, a deaminase domain, and a UGI can be fused to any combination of peptide markers and affinity polypeptides as described in this document, thereby recruiting the deaminase domain and the UGI to the CRISPR-Cas effector polypeptide and a target nucleic acid. In some embodiments, a guide nucleic acid can be linked to a recruitment RNA motif and one or more deaminase and / or UGI domains can be fused to an affinity polypeptide that is capable of interacting with the recruitment RNA motif, thereby recruiting the deaminase domain and UGI to a target nucleic acid.

[00273] A useful uracil glycosylase inhibitor with the invention can be any protein that is capable of inhibiting a base excision repair enzyme of uracil-DNA glycosylase. In some embodiments, a UGI domain comprises a wild-type UGI or a fragment thereof. In some embodiments, a useful UGI domain with the invention can be about 70% to about 100% identical (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%, 99.5%, or 100% identical, and any range or value therein) to the amino acid sequence of a naturally occurring UGI domain. In some embodiments, a UGI domain may comprise the amino acid sequence of SEQ ID NO:41 or a polypeptide with about 70% to about 99.5% sequence identity with the amino acid sequence of SEQ ID NO:41 (e.g., at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of SEQ ID NO:41). For example, Petition 870250103857, dated 11 / 13 / 2025, pp. 147 / 212 138 / 166 In some embodiments, a UGI domain may comprise a fragment of the amino acid sequence of SEQ ID NO:41 that is 100% identical to a portion of consecutive nucleotides (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 consecutive nucleotides; e.g., approximately 10, 15, 20, 25, 30, 35, 40, 45, to approximately 50, 55, 60, 65, 70, 75, 80 consecutive nucleotides) of the amino acid sequence of SEQ ID NO:41. In some embodiments, a UGI domain may be a variant of a known UGI (e.g., SEQ ID NO: 41) having approximately 70% to approximately 99.5% sequence identity (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%, 99.5% sequence identity and any range or value contained therein) to the known UGI.In some embodiments, a polynucleotide encoding a UGI can be codon-optimized for expression in a plant (e.g., a plant), and the codon-optimized polypeptide can be about 70% to about 99.5% identical to the reference polynucleotide.

[00274] A useful adenine deaminase (or adenosine deaminase) with this invention may be any known or subsequently identified adenine deaminase from any organism (see, for example, U.S. Patent No. 10,113,163, which is incorporated by reference herein for its disclosure of adenine deaminases). An adenine deaminase may catalyze the hydrolytic deamination of adenine or adenosine. In some embodiments, the adenine deaminase may catalyze the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In some embodiments, the adenosine deaminase may catalyze the hydrolytic deamination of adenine or adenosine in DNA. In some embodiments, an adenine deaminase encoded by a construct Petition 870250103857, dated 11 / 13 / 2025, pages 148 / 212 139 / 166 of nucleic acid of the invention can generate an A^G conversion on the sense strand (e.g., +; template) of the target nucleic acid or a T^C conversion on the antisense strand (e.g., -, complementary) of the target nucleic acid.

[00275] In some embodiments, an adenosine deaminase may be a variant of a natural adenine deaminase. Thus, in some embodiments, an adenosine deaminase may be about 70% to 100% identical to a wild-type adenine deaminase (for example, 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 100% identical, and any range or value therein contained, to a naturally occurring adenine deaminase). In some embodiments, the deaminase or deaminase does not occur in nature and may be referred to as an engineered, mutated, or evolved adenosine deaminase. Thus, for example, a modified, mutated, or evolved adenine deaminase polypeptide or adenine deaminase domain may be about 70% to 99.9% identical to a naturally occurring adenine deaminase polypeptide / domain (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%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7% 99.8% or 99.9% identical, and any range or value therein, to a naturally occurring adenine deaminase polypeptide or adenine deaminase domain). In some embodiments, the adenosine deaminase may be from a bacterium (e.g., Escherichia coli, Staphylococcus aureus, Haemophilus influenzae, Caulobacter crescentus, and the like). In some embodiments, a polynucleotide encoding an adenine deaminase polypeptide / domain may have a codon optimized for expression in a plant. Petition 870250103857, dated 11 / 13 / 2025, pp. 149 / 212 140 / 166

[00276] In some embodiments, an adenine deaminase domain may be a wild-type tRNA-specific adenosine deaminase domain, for example, a tRNA-specific adenosine deaminase (TadA) and / or a mutated / evolved adenosine deaminase domain, for example, a mutated / evolved tRNA-specific adenosine deaminase domain (TadA*). In some embodiments, a TadA domain may be from E. coli. In some embodiments, TadA may be modified, for example, truncated, lacking one or more N-terminal and / or C-terminal amino acids relative to a full-length TadA (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal and / or C-terminal amino acid residues may be absent relative to a full-length TadA). In some embodiments, a TadA polypeptide or TadA domain does not comprise an N-terminal methionine. In some embodiments, a wild-type E.E. coli TadA comprises the amino acid sequence of SEQ ID NO:30. In some embodiments, a mutant / evolved E. coli TadA* comprises the amino acid sequence of SEQ ID NOs:31-40 (e.g., SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40). In some embodiments, a polynucleotide encoding a TadA / TadA* may have codon-optimized for expression in a plant.

[00277] A cytosine deaminase catalyzes the deamination of cytosine and results in a thymidine (via a uracil intermediate), causing a C to T conversion, or a G to A conversion on the complementary strand of the genome. Thus, in some embodiments, the cytosine deaminase encoded by the polynucleotide of the invention generates a C^T conversion on the sense strand (e.g., +; template) of the target nucleic acid or a G^A conversion on the antisense strand (e.g., -, complementary) of the target nucleic acid.

[00278] In some forms, adenine deaminase encodes Petition 870250103857, dated 11 / 13 / 2025, pp. 150 / 212 141 / 166 of the nucleic acid construct of the invention generates an A>G conversion on the sense strand (e.g., +; template) of the target nucleic acid or a T>C conversion on the antisense strand (e.g., -, complementary) of the target nucleic acid.

[00279] The nucleic acid constructs of the invention encoding a base editor comprising a sequence-specific nucleic acid binding protein and a cytosine deaminase polypeptide, and nucleic acid constructs / expression cassettes / vectors encoding the same, can be used in combination with guide nucleic acids to modify the target nucleic acid, including, but not limited to, generating C>T or G>A mutations in a target nucleic acid, including, but not limited to, a plasmid sequence; generating C>T or G>A mutations in a coding sequence to alter an amino acid identity; generating C>T or G>A mutations in a coding sequence to generate a stop codon; generating C>T or G>A mutations in a coding sequence to interrupt a start codon; generating point mutations in genomic DNA to disrupt function;and / or generation of point mutations in genomic DNA to disrupt splicing junctions.

[00280] The nucleic acid constructs of the invention encoding a base editor comprising a sequence-specific nucleic acid binding protein and an adenine deaminase polypeptide, and expression cassettes and / or vectors encoding the same, can be used in combination with guide nucleic acids to modify a target nucleic acid, including, but not limited to, generating A>G or T>C mutations in a target nucleic acid, including, but not limited to, a plasmid sequence; generating A>G or T>C mutations in a coding sequence to alter an amino acid identity; generating A>G or T>C mutations. Petition 870250103857, dated 11 / 13 / 2025, pages 151 / 212 142 / 166 T^C mutations in a coding sequence to generate a stop codon; generation of A^G or T^C mutations in a coding sequence to interrupt a start codon; generation of point mutations in genomic DNA to disrupt function; and / or generation of point mutations in genomic DNA to disrupt splice junctions.

[00281] The nucleic acid constructs of the invention comprising a CRISPR-Cas effector protein or a fusion protein thereof can be used in combination with a guide RNA (gRNA, CRISPR matrix, CRISPR RNA, crRNA), designed to function with the effector protein or encoded CRISPR-Cas domain, to modify a target nucleic acid. A useful guide nucleic acid with this invention comprises at least one spacer sequence and at least one repeat sequence.The guide nucleic acid is capable of forming a complex with the CRISPR-Cas nuclease domain encoded and expressed by a nucleic acid construct of the invention, and the spacer sequence is capable of hybridizing with a target nucleic acid, thus guiding the complex (e.g., a CRISPR-Cas effector fusion protein (e.g., CRISPR-Cas effector domain fused to a deaminase domain and / or a CRISPR-Cas effector domain fused to a peptide marker or an affinity polypeptide to recruit a deaminase domain and, optionally, a UGI) to the target nucleic acid, wherein the target nucleic acid can be modified (e.g., cleaved or edited) or modulated (e.g., by modulating transcription) by the deaminase domain.

[00282] As an example, a nucleic acid construct encoding a Cas9 domain linked to a cytosine deaminase domain (e.g., fusion protein) can be used in combination with a Cas9 guide nucleic acid to modify a target nucleic acid, where the cytosine deaminase domain of the fusion protein deaminates. Petition 870250103857, dated 11 / 13 / 2025, pages 152 / 212 143 / 166 a cytosine base in the target nucleic acid, thus editing the target nucleic acid. In another example, a nucleic acid construct encoding a Cas9 domain linked to an adenine deaminase domain (e.g., fusion protein) can be used in combination with a Cas9 guide nucleic acid to modify a target nucleic acid, where the adenine deaminase domain of the fusion protein deaminates an adenosine base in the target nucleic acid, thus editing the target nucleic acid.

[00283] Similarly, a nucleic acid construct encoding a Cas12a domain (or another selected CRISPR-Cas nuclease, for example, C2c1, C2c3, Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Cas1, Cas1B, Cas2, Cas3, Cas3', Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG) and / or Csf5) linked to a cytosine deaminase domain or adenine deaminase domain (e.g., fusion protein) can be used in combination with a Cas12a guide nucleic acid (or the guide nucleic acid for another selected CRISPR-Cas nuclease) to modify a target nucleic acid, wherein the cytosine deaminase domain or adenine deaminase domain of the fusion protein deaminates a cytosine base in the target nucleic acid.editing the target nucleic acid.

[00284] A guide nucleic acid, guide RNA, gRNA, CRISPR RNA / DNA, crRNA or crDNA, as used herein, means a nucleic acid comprising at least one spacer sequence, which is complementary to (and hybridizes with) a target DNA (e.g., protospacer) and at least one repeat sequence (e.g., a repeat of a CRISPR-Cas Type V Cas12a system, or a fraction of a spacer sequence). Petition 870250103857, dated 11 / 13 / 2025, pp. 153 / 212 144 / 166 a segment or portion thereof; a repeat of a CRISPR-Cas Type II Cas9 system, or a fragment thereof; a repeat of a CRISPR-Cas Type V C2c1 system, or a fragment thereof; a repetition of a CRISPR-Cas system of, for example, C2c3, Cas12a (also known as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Cas1, Cas1B, Cas2, Cas3, Cas3', Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG) and / or Csf5, or a fragment thereof), wherein the repeat sequence may be attached to the 5' end and / or the 3' end of the spacer sequence. 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.

[00285] In some embodiments, a Cas12a gRNA may comprise, from 5' to 3', a repeat sequence (complete or part thereof (loop); for example, pseudonode-like structure) and a spacer sequence.

[00286] In some embodiments, a guide nucleic acid may comprise more than one repeat-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-repeat-spacer and the like). The guide nucleic acids of this invention are synthetic, man-made, and not found in nature. A gRNA may be quite long and may be used as an aptamer (as in the MS2 recruitment strategy) or other RNA structures hanging from the spacer. Petition 870250103857, dated 11 / 13 / 2025, pages 154 / 212 145 / 166

[00287] A repeat sequence, as used in this document, refers, for example, to any repeat sequence of a wild-type CRISPR Cas locus (e.g., a Cas9 locus, a Cas12a locus, a C2c1 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 useful repeat sequence with this invention may be any known or subsequently identified repeat sequence of a CRISPR-Cas locus (e.g., Type I, Type II, Type III, Type IV, Type V, or Type VI) or may 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., loop).Thus, in some embodiments, a repeat sequence may be identical or substantially identical to a repeat sequence of wild-type CRISPR-Cas Type I loci, CRISPR-Cas Type II loci, CRISPR-Cas Type III loci, CRISPR-Cas Type IV loci, CRISPR-Cas Type V loci, and / or CRISPR-Cas Type VI loci. A repeat sequence of a wild-type CRISPR-Cas locus can be determined by means of established algorithms, such as the use of the CRISPRfinder offered by CRISPRdb (see Grissa et al. Nucleic Acids Res. 35(Web Server issue):W52-7). In some embodiments, a repeat sequence or part thereof is linked at its 3' end to the 5' end of a spacer sequence, thus forming a repeat spacer sequence (e.g., guide nucleic acid, guide RNA / DNA, crRNA, crDNA).

[00288] In some forms, a repeating sequence comprises, consists essentially of, or consists of at least Petition 870250103857, dated 11 / 13 / 2025, pp. 155 / 212 146 / 166 nucleotides depending on the specific repeat and whether the guide nucleic acid comprising the repeat is processed or unprocessed (e.g., approximately 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 contained therein). In some embodiments, a repeating 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.

[00289] A repeat sequence attached to the 5' end of a spacer sequence may comprise a portion of a repeat sequence (for example, 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 attached to the 5' end of a spacer sequence may be about five to about ten consecutive nucleotides long (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 (e.g., 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100%)) with the same region (e.g., 5' end) of a wild-type CRISPR Cas repeat nucleotide sequence.In some modalities, a part of a repeating sequence may comprise a similar structure. Petition 870250103857, dated 11 / 13 / 2025, pages 156 / 212 147 / 166 to a pseudonode at its 5' end (e.g., handle).

[00290] 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) (e.g., a portion of consecutive nucleotides of a sequence that (a) comprises a nucleotide sequence with at least 80% sequence identity (e.g., at least approximately 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity) for any of the SEQ ID NOs: 69, 70, 75, 76, 128, 129 or 162-173; at least 80% sequence identity with a sequence of nucleotides from any of the SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134; (c) encodes an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 71, 77, 130, or 174-182; and / or (d) encodes a region of an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 126, 127, 136 or 137, optionally where the sequence identity of (a), (b), (c) and / or (d) may be at least 85%, or at least 90%, or may be at least 95%, optionally the sequence identity may be 100%. In some embodiments, a spacer sequence (e.g., one or more spacers) may include, but is not limited to, the nucleotide sequence of any of the SEQ IDs NOS: 138-142, or any combination thereof.The spacing 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%,). Petition 870250103857, dated 11 / 13 / 2025, pp. 157 / 212 148 / 166 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100%)) for a target nucleic acid. Thus, in some embodiments, the spacer sequence may have one, two, three, four, or five mismatches compared to the target nucleic acid, mismatches that may be contiguous or non-contiguous. In some embodiments, the spacer sequence may have 70% complementarity with a target nucleic acid. In other embodiments, the spacer nucleotide sequence may have 80% complementarity with a target nucleic acid. In still other embodiments, the spacer nucleotide sequence may have 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% complementarity, and similar, with the target nucleic acid (protospacer). In some embodiments, the spacer sequence is 100% complementary to the target nucleic acid.A spacer sequence can have a length of about 15 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 contained therein). Thus, in some 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 to about 30 nucleotides long. In some embodiments, the spacer is about 20 nucleotides long. In some embodiments, the spacer is about 21, 22, or 23 nucleotides long. In some embodiments, a spacer sequence may comprise the sequence SEQ ID NOs:138-142, or its reverse complement.

[00291] In some embodiments, the 5' region of a guide nucleic acid spacer sequence may be identical to a target DNA, while the 3' region of the spacer may be substantially different. Petition 870250103857, dated 11 / 13 / 2025, pp. 158 / 212 149 / 166 complementary to the target DNA (see, for example, a spacer sequence of a Type V CRISPR-Cas system), or the 3' region of a guide nucleic acid spacer sequence may be identical to a target DNA, while the 5' region of the spacer may be substantially complementary to the target DNA (see, for example, a spacer sequence of a Type II CRISPR-Cas system), and therefore the overall complementarity of the spacer sequence to the target DNA may be less than 100%. Thus, for example, in a guide for a Type V CRISPR-Cas system, the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides in the 5' region (i.e., seed region) of, for example, a 20-nucleotide spacer sequence may be 100% complementary to the target DNA, while the remaining nucleotides in the 3' region of the spacer sequence are substantially complementary (i.e., at least about 70% complementary) to the target DNA.In some embodiments, the first 1 to 8 nucleotides (e.g., the first 1, 2, 3, 4, 5, 6, 7, 8 nucleotides and any gaps therein) of the 5' end of the spacer sequence may be 100% complementary to the target DNA, 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) for the target DNA.

[00292] As another example, in a guide 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 DNA, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary (by Petition 870250103857, dated 11 / 13 / 2025, pages 159 / 212 150 / 166 example, at least about 70% complementary) to the target DNA. 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 gaps therein) of the 3' end of the spacer sequence may be 100% complementary to the target DNA, while the remaining nucleotides in the 5' region of the spacer sequence are substantially 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, for the target DNA.

[00293] In some embodiments, a seed region of a spacer may be about 8 to about 10 nucleotides long, about 5 to about 6 nucleotides long, or about 6 nucleotides long.

[00294] As used herein, a target nucleic acid, target DNA, target nucleotide sequence, target region, or a target region in the genome refers to a region of a plant genome 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 of this invention. A useful target region for a CRISPR-Cas system can be located immediately 3' (e.g., CRISPR-Cas Type V system) or immediately 5' (e.g., CRISPR-Cas Type II system) to a PAM sequence in the organism's genome (e.g., a plant genome). A target region can be selected from any region. Petition 870250103857, dated 11 / 13 / 2025, pages 160 / 212 151 / 166 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.

[00295] A protospacer sequence refers to the target double-stranded DNA and specifically to the portion of the target DNA (e.g., or target region in the genome) that is wholly or substantially complementary to (and hybridizes with) the spacer sequence of CRISPR repeat spacer sequences (e.g., guide nucleic acids, CRISPR arrays, crRNAs).

[00296] In the case of CRISPR-Cas Type V (e.g., Cas12a) and CRISPR-Cas Type II (Cas9) systems, the protospacer sequence is flanked by (e.g., immediately adjacent to) a protospacer adjacent motif (PAM). For CRISPR-Cas Type IV systems, the PAM is located at the 5' end of the non-target strand and at the 3' end of the target strand (see below for an example). 5'-NNNNNNNNNNNNNNNNNNN-3' RNA Spacer IIIIIIIIIIIIIIIIIII 3'-AAANNNNNNNNNNNNNNNNNNNN-5' Target Tape III 5'-TTTNNNNNNNNNNNNNNNNNNN-3' Non-target tape

[00297] In the case of CRISPR-Cas Type II systems (e.g., Cas9), the PAM is located immediately 3' from the target region. The PAM for CRISPR-Cas Type I systems is located 5' from the target strand. There is no known PAM for CRISPR-Cas Type III systems. Makarova et al. describes the nomenclature for all classes, types, and subtypes of CRISPR systems (Nature Reviews Microbiology 13:722-736 (2015)). Guide structures and PAMs are described by R. Barrangou (Genome Biol. 16:247 (2015)).

[00298] Canonical Cas12a PAMs are T-rich. In some embodiments, a canonical Cas12a PAM sequence may be 5'-TTN, 5'-TTTN, or 5'-TTTV. In some embodiments, canonical Cas9 PAMs (e.g., S. pyogenes) may be 5'-NGG-3'. In some Petition 870250103857, dated 11 / 13 / 2025, pages 161 / 212 152 / 166 modalities, non-canonical PAMs can be used, but may be less efficient.

[00299] Additional PAM sequences can be determined by those skilled in the art through established experimental and computational approaches. Thus, for example, experimental approaches include targeting a sequence flanked by all possible nucleotide sequences and identifying sequence members that do not pass targeting, such as through transformation of the target plasmid DNA (Esvelt et al. 2013. Nat. Methods 10:11161121; Jiang et al. 2013. Nat. Biotechnol. 31:233-239). In some respects, a computational approach may 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 and Barrangou. 2014. Appl. Environ. Microbiol. 80:994-1001; Mojica et al. 2009. Microbiology 155:733-740).

[00300] In some embodiments, the present invention provides expression cassettes and / or vectors comprising the nucleic acid constructs of the invention (for example, one or more components of an editing system of the invention). In some embodiments, expression cassettes and / or vectors comprising the nucleic acid constructs of the invention and / or one or more guide nucleic acids may be provided. In some embodiments, a nucleic acid construct of the invention encoding a base editor (for example, a construct comprising a CRISPR-Cas effector protein and a deaminase domain (for example, a fusion protein)) or the components for base editing (for example, a CRISPR-Cas effector protein fused to a peptide marker or an affinity polypeptide, a deaminase domain fused to a peptide marker or an affinity polypeptide, and / or a UGI fused to a marker) Petition 870250103857, dated 11 / 13 / 2025, pages 162 / 212 153 / 166 peptide or an affinity polypeptide), may be comprised in the same or in a separate expression cassette or vector from that comprising one or more guide nucleic acids. When the nucleic acid construct encoding a base editor or the components for base editing are comprised in separate expression cassettes or vectors from that comprising the guide nucleic acid, a target nucleic acid may be contacted with (e.g., provided with) the expression cassettes or vectors encoding the base editor or components for base editing in any order relative to each other and the guide nucleic acid, for example, before, simultaneously with, or after the expression cassette comprising the guide nucleic acid is provided (e.g., in contact with the target nucleic acid).

[00301] The fusion proteins of the invention may comprise sequence-specific nucleic acid binding domains (e.g., sequence-specific DNA binding domains), CRISPR-Cas polypeptides and / or deaminase domains fused to peptide markers or affinity polypeptides that interact with peptide markers, as known in the art, for use in recruiting deaminase to the target nucleic acid. The recruitment methods may also comprise guide nucleic acids linked to RNA recruitment motifs and deaminases fused to affinity polypeptides capable of interacting with RNA recruitment motifs, thereby recruiting deaminase to the target nucleic acid. Alternatively, chemical interactions may be used to recruit polypeptides (e.g., deaminases) to a target nucleic acid.

[00302] A useful peptide tag (e.g., epitope) with this invention may include, but is not limited to, a GCN4 peptide tag (e.g., Sun-Tag), a c-Myc affinity tag, an HA affinity tag, a His affinity tag, a tag Petition 870250103857, dated 11 / 13 / 2025, pages 163 / 212 154 / 166 affinity S tag, a methionine-His affinity tag, an RGD-His affinity tag, a FLAG octapeptide, a streptococcal tag or streptococcal II tag, a V5 tag and / or a VSV-G epitope. Any epitope that can be linked to a polypeptide and for which there is a corresponding affinity polypeptide that can be linked to another polypeptide can be used with this invention as a peptide marker. In some embodiments, a peptide marker may comprise 1 or 2 or more copies of a peptide marker (e.g., repeat unit, multimerized epitope (e.g., tandem repeats)) (e.g., 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 or more repeat units). In some embodiments, an affinity polypeptide that interacts with / binds to a peptide marker may be an antibody.In some embodiments, an affinity polypeptide that binds to a peptide marker may be synthetic (e.g., evolved by affinity interaction), including, without limitation, an affibody, an anticalin, a monobody, and / or a DARPin (see, for example, Sha et al., Protein Sci. 26(5):910-924 (2017)); Gilbreth (Curr Opin Struc Biol 22(4):413-420 (2013)), U.S. Patent No. 9,982,053, each of which is incorporated by reference in its entirety for the teachings relevant to affibodies, anticalins, monobodies, and / or DARPins. Examples of peptide marker sequences and their affinity polypeptides include, without limitation, the amino acid sequences of SEQ ID Nos:42-44.

[00303] In some embodiments, a guide nucleic acid can be linked to an RNA recruitment motif, and a polypeptide to be recruited (e.g., a deaminase) can be fused to an affinity polypeptide that binds to the RNA recruitment motif, wherein the guide binds to the target nucleic acid and the recruitment motif binds to the target nucleic acid. Petition 870250103857, dated 11 / 13 / 2025, pp. 164 / 212 155 / 166 RNA binds to the affinity polypeptide, thus recruiting the polypeptide to the guide and bringing the target nucleic acid into contact with the polypeptide (e.g., deaminase). In some embodiments, two or more polypeptides can be recruited to a guide nucleic acid, thus bringing the target nucleic acid into contact with two or more polypeptides (e.g., deaminases). Examples of RNA recruitment motifs and their affinity polypeptides include, among others, sequences with SEQ IDs NOS:45-55.

[00304] In some embodiments, a polypeptide fused to an affinity polypeptide may be a reverse transcriptase, and the guide nucleic acid may be an extended guide nucleic acid linked to an RNA recruitment motif. In some embodiments, an RNA recruitment motif may be located at the 3' end of the extended portion of an extended guide nucleic acid (e.g., 5'-3', extended repeat spacer portion (RT template primer binding site)-RNA recruitment motif). In some embodiments, an RNA recruitment motif may be incorporated into the extended portion.

[00305] In some embodiments of the invention, an extended guide RNA and / or guide RNA may be linked to one or two or more RNA recruitment motifs (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more motifs; for example, at least 10 to about 25 motifs), optionally where the two or more RNA recruitment motifs may be the same RNA recruitment motif or different RNA recruitment motifs. In some embodiments, an RNA recruitment motif and the corresponding affinity polypeptide may include, without limitation, a Ku telomerase binding motif (e.g., Ku-binding clamp) and the corresponding Ku affinity polypeptide (e.g., Ku heterodimer), an Sm7 telomerase binding motif and the corresponding affinity polypeptide Petition 870250103857, dated 11 / 13 / 2025, pp. 165 / 212 156 / 166 respondent© Sm7, an MS2 stem-loop phage operator and the corresponding affinity polypeptide MS2 Coating Protein (MCP), a PP7 stem-loop phage operator and the corresponding affinity polypeptide PP7 Coating Protein (PCP), an SfMu Com stem-loop phage and the corresponding affinity polypeptide RNA-binding Protein, a PUF (PBS) binding site and the Pumilio / fem-3 mRNA binding factor (PUF) affinity polypeptide, and / or a synthetic RNA-aptamer and aptamer ligand as the corresponding affinity polypeptide. In some embodiments, the RNA recruitment motif and the corresponding affinity polypeptide may be an MS2 stem-loop phage operator and the MS2 coat protein (MCP) affinity polypeptide.In some embodiments, the RNA recruitment motif and the corresponding affinity polypeptide may be a PUF binding site (PBS) and the Pumilio / fem-3 mRNA binding factor (PUF) affinity polypeptide.

[00306] In some embodiments, the components for recruiting polypeptides and nucleic acids may be those that function through chemical interactions which may include, but are not limited to, rapamycin-inducible dimerization of FRB FKBP; biotin-streptavidin; SNAP tag; Halo tag; CLIP tag; compound-induced DmrA-DmrC heterodimer; bifunctional ligand (e.g., fusion of two protein-binding chemicals, e.g., dihydrofolate reductase (DHFR)).

[00307] In some embodiments, the nucleic acid constructs, expression cassettes, or vectors of the invention that are optimized for expression in a plant may be about 70% to 100% identical (for example, 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%, 99.5% Petition 870250103857, dated 11 / 13 / 2025, pages 166 / 212 157 / 166 or 100%) to nucleic acid constructs, expression cassettes, or vectors comprising the same polynucleotide(s) but not codon-optimized for expression in a plant.

[00308] In addition, cells comprising one or more polynucleotides, guide nucleic acids, nucleic acid constructs, expression cassettes or vectors of the invention are provided herein.

[00309] The nucleic acid constructs of the invention (for example, a construct comprising a sequence-specific DNA binding domain, a CRISPR-Cas effector domain, a deaminase domain, reverse transcriptase (RT), RT template and / or a guide nucleic acid, etc.) and expression cassettes / vectors comprising them can be used as an editing system of this invention to modify target nucleic acids and / or their expression.

[00310] A functional target nucleic acid WOX (e.g., that is specifically expressed in tissues that give rise to trichomes, spines and / or prickles; e.g., that is expressed in a tissue-specific manner in vascular tissue and / or epidermal tissue) present in any Rosaceae plant or plant part may be modified (e.g., mutated, e.g., base-edited, cleaved, cut, etc.) as described herein to modify the architecture of spines and / or prickles, to reduce the number and / or size of spines and / or prickles, and / or eliminate spines and / or prickles using the polypeptides, polynucleotides, ribonucleoproteins (RNPs), nucleic acid constructs, expression cassettes and / or vectors of the invention. Non-limiting examples of Rosaceae plant species that may be modified as described herein may include, but are not limited to, Rosa spp., Hulthemia spp., Hesperrhodos spp., Platyrhodon spp., or Rubus spp.In some variations, the Rubus plant may be a mulberry plant, a black raspberry plant, or a red raspberry plant. In some variations... Petition 870250103857, dated 11 / 13 / 2025, pp. 167 / 212 158 / 166 dades, Rubus spp. can be blackberry or black raspberry, optionally, wherein the Rubus spp. plant is Rubus allegheniensis (Allegheny blackberry), Rubus armeniacus (Himalayan blackberry), Rubus canadensis (Canadian blackberry), Rubus fruticosus agg. (European blackberry), Rubus laciniatus (perennial cut-leaf blackberry), Rubus pensilvanicus (Pennsylvania blackberry), Rubus trifidus (Japanese blackberry), Rubus ursinus (wild blackberry on the right), Rubus hybrid (e.g., progeny resulting from the hybridization of two or more of the previous Rubus species), Rubus leucodermis (white-skinned raspberry or western black raspberry) or Rubus occidentale (black raspberry).In some embodiments, Rubus spp. may be a raspberry plant (red raspberry), including, but not limited to, Rubus crataegifolius (Asian raspberry), Rubus gunnianus (Tasmanian alpine raspberry), Rubus idaeus (European red raspberry), Rubus parvifolius (Australian native raspberry), Rubus phoenicolasius (wine raspberry), Rubus rosifolius (Mauritius raspberry), Rubus strigosus (American red raspberry) (syn. R. idaeus var. strigosus), or Rubus ellipticus (Himalayan yellow raspberry). In some embodiments, Rosa spp. is a rose plant and / or Rosa spp. is Rosa chinensis.

[00311] 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 buds, ovules, seeds, embryos, nuts, grains, ears, spikes, and husks); vegetative tissues (e.g., petioles, stems, roots, root hairs, root tips, pith, coleoptiles, stalks, buds, 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, stomata, Petition 870250103857, dated 11 / 13 / 2025, pages 168 / 212 159 / 166 guard cells, cuticle, mesophyll cells; callus tissue; and cuttings. The term plant part also includes plant cells, including plant cells that are intact in plants and / or plant parts, plant protoplasts, plant tissues, plant organs, plant cell tissue cultures, plant calluses, plant clumps, and the like. As used herein, sprout refers to the above-ground parts, including leaves and stems. As used herein, the term tissue culture encompasses tissue cultures, cells, protoplasts, and calluses.

[00312] As used herein, plant cell refers to a structural and physiological unit of the plant, which normally comprises a cell wall, but also includes protoplasts. A plant cell of the present invention may be in the form of a single isolated cell or may be a cultured cell or may be part of a higher organized unit, such as, for example, a plant tissue (including callus) or a plant organ. In some embodiments, a plant cell may be an algal cell. A protoplast is an isolated 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 may be plant germplasm. In some aspects, a plant cell may be a non-propagating plant cell that does not regenerate into a plant.

[00313] “Plant cell culture” means cultures of plant units, such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, sacs Petition 870250103857, dated 11 / 13 / 2025, pages 169 / 212 160 / 166 embryonic cells, zygotes and embryos at various stages of development.

[00314] As used herein, a plant organ is a distinct, visibly structured and differentiated part of a plant, such as a root, stem, leaf, flower bud or embryo.

[00315] Plant tissue, as used herein, means a group of plant cells organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, among others, whole plants, plant organs, plant seeds, tissue culture, and any groups of plant cells organized into structural and / or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue, as listed above or otherwise encompassed by this definition, is not intended to be exclusive of any other type of plant tissue.

[00316] In some embodiments of the invention, a transgenic tissue culture or transgenic plant cell culture is provided, wherein the transgenic tissue or cell culture comprises a nucleic acid molecule / nucleotide sequence of the invention. In some embodiments, transgenes can be eliminated from a plant developed from the transgenic tissue or cell by crossing the transgenic plant with a non-transgenic plant and selecting from the progeny of the plants that comprise the desired gene edit and not the transgenes used in producing the edit.

[00317] The invention will now be described with reference to the following examples. It should be understood that these examples are not intended to limit the scope of the invention claims, but rather to be exemplary of certain embodiments. Any variations in the methods exemplified that occur to those skilled in the art must be within Petition 870250103857, dated 11 / 13 / 2025, pages 170 / 212 161 / 166 of the scope of the invention. EXAMPLES Example 1. Editing strategy

[00318] A strategy for generating knock-out or knockdown edits in the blackberry WOX1 gene (SEQ ID NO: 69) was developed to alter thorn formation in any part of the plant, including stem and leaves. The blackberry variety employed in this example is tetraploid and contains two WOX1 genes that were found to be homozygous at one genetic locus and heterozygous at the second locus. One of the four copies of the WOX1 gene comprises the nucleic acid sequence of SEQ ID NO: 69, and the remaining three copies of the WOX1 gene comprise the nucleic acid sequence of SEQ ID NO: 72. Copies of WOX1 genes with the nucleic acid sequence of SEQ ID NO: 72 were found to contain a polymorphism that leads to a non-functional protein. Based on this analysis, only the WOX1 gene of SEQ ID NO: 69 was considered a functional copy of the WOX1 gene in the blackberry variety that was selected for editing, as described in this example.To generate a range of alleles of SEQ ID NO: 69, multiple CRISPR guide nucleic acids comprising spacers (SEQ ID NOs: 141-142) having complementarity with the 3' end of exon 1 within the WOX1 gene of SEQ ID NO: 69 were designed and placed in the pWISE8806 construct.

[00319] Lines carrying edits in the WOX1 genes were screened and those that showed approximately 10% of sequencing reads with edits in the target gene were advanced to the next generation. Example 2: Edited alleles of WOX1

[00320] Blackberry plants with edited alleles of the WOX1 gene were generated as described in Example 1 and are described in more detail in Table 2. Petition 870250103857, dated 11 / 13 / 2025, pages 171 / 212 162 / 166

[00321] Plants generated as described in Example 1 were transferred from tissue culture to buffer media and then to a greenhouse environment. The plants were inspected for the development of spines and glandular trichomes one month after transfer from tissue culture. At this stage of their growth, the plants were sufficiently developed that, in unedited plants (or plants that included a functional copy of the W0X1 gene), spines were apparent on the cane, as well as on the petioles and midrib of the leaves.

[00322] The edited W0X1 alleles generated in Example 1 were clustered at the 3' end of exon 1, and all edits were deletions. In some of the lines, the deletion is predicted to affect the exon 1:intron 1 splice site. While we do not wish to be bound by any theory, it is expected that in alleles where the intron 1 splice site has been altered, intron 1 is deleted using a secondary splice site. Note that out-of-frame deletions rendered the functional copy of W0X1 non-functional. Plants comprising these out-of-frame edits exhibited a spineless phenotype. In-frame deletions (including those affecting the intron 1 splice site) did not alter W0X1 activity; that is, the functional copy remained functional, and therefore spines were present in these plants. Table 2: Edited mulberry plants generated with pWISE8806 Plant Name Phenotype Edition Description Comments CE196774 Spineless 10 bp deletion (CAGCATCAGG; SEQ ID NO: 157) starting at position 2626 of SEQ ID NO: 69 giving rise to the genomic sequence of SEQ ID NO: 143 Out-of-frame mutation that also removes the splice site at the 3' end of exon 1 and leads to an early stop codon. Petition 870250103857, dated 11 / 13 / 2025, pp. 172 / 212 163 / 166 Plant Name Phenotype Edition Description Comments CE196763 Spineless 6 bp deletion (CAGGGT) starting at position 2632 of SEQ ID NO: 69 giving rise to the genomic sequence of SEQ ID NO: 144 Out-of-frame mutation that also removes the splice site at the 3' end of exon 1 and leads to an early stop codon. CE196749 Spiny Deletion of 3 bp (CAG) starting at position 2632 of SEQ ID NO: 69 Deletion in frame CE196760 Spiny Deletion of 6 bp (CATCAG) starting at position 2629 of SEQ ID NO: 69 Deletion in frame CE196780 Spiny Deletion of 10 bp (GCATCAGGGT; SEQ ID NO: 158) starting at position 2628 of SEQ ID NO: 69 In the frame deletion, this also removes the splice site at the 3' end of exon 1. CE196762 Spiny Deletion of 9 bp (CCAGCATCA) starting at position 2625 of SEQ ID NO: 69 Deletion in frame CE196764 Spiny Deletion of 3 bp (TCA) starting at position 2631 of SEQ ID NO: 69 Deletion in Example 3: Knock-down editing strategy

[00323] A strategy to generate knock-out or knockdown edits in the blackberry gene W0X1 (SEQ ID NO: 69) was developed to alter thorn formation in any part of the plant, including stem and leaves. The blackberry variety employed in this example is tetraploid and contains two W0X1 genes that were considered ho Petition 870250103857, dated 11 / 13 / 2025, pp. 173 / 212 164 / 166 monozygotes at one genetic locus and heterozygotes at the second locus. One of the four copies of the WOX1 gene comprises the nucleic acid sequence SEQ ID NO: 69, and the remaining three copies of the WOX1 gene comprise the nucleic acid sequence SEQ ID NO: 72. Copies of WOX1 genes with the nucleic acid sequence SEQ ID NO: 72 were found to contain a polymorphism leading to a non-functional protein. To generate a range of alleles of SEQ ID NO: 69, multiple CRISPR guide nucleic acids comprising spacers (SEQ ID NOs: 138-140) having complementarity to the homeodomain within the WOX1 gene of SEQ ID NO: 69 were designed and placed in the pWISE8807 construct.

[00324] Lines carrying edits in the WOX1 genes were screened and those that showed approximately 10% of sequencing reads with edits in the target gene were advanced to the next generation. Example 4: Edited alleles of WOX1

[00325] Blackberry plants with edited alleles of the WOX1 gene were generated as described in Example 3 and are described in more detail in Table 3.

[00326] Plants generated as described in Example 3 were transferred from tissue culture to buffer media and then to a greenhouse environment. The plants were inspected for the development of spines and glandular trichomes one month after transfer from tissue culture. At this growth stage, the plants were sufficiently developed such that, in unedited plants (or plants including a functional copy of the WOX1 gene), spines were apparent on the stem, as well as on the petioles and midrib of the leaves.

[00327] The edited WOX1 alleles generated in Example 3 were clustered in exon 2 in the homeodomain region of the WOX1 protein. Petition 870250103857, dated 11 / 13 / 2025, pages 174 / 212 165 / 166 Table 3: Edited blackberry plants generated with pWISE8807 Plant Name Phenotype Edition Description Comments CE197153 Spineless 9 bp deletion (CATCAAGAC) starting at position 2918 of SEQ ID NO: 69; and 1 bp deletion (C) at position 2961 of SEQ ID NO: 69 resulting in the genomic sequence of SEQ ID NO: 145 Out-of-frame mutation leading to an early stop codon and a protein with the amino acid sequence of SEQ ID NO: 146 CE197154 Spineless 6 bp deletion (ATCAAG) starting at position 2919 of SEQ ID NO: 69; and 9 bp deletion (CGGCGCTGC) starting at position 2956 of SEQ ID NO: 69; and a 7 bp deletion (GGTTTCA) starting at position 3007 of SEQ ID NO: 69 giving rise to the genomic sequence of SEQ ID NO: 147. Out-of-frame mutation leading to an early stop codon and a protein with the amino acid sequence of SEQ ID NO: 148 CE197157. No spikes. 2 bp deletion (GC) starting at position 2958 of SEQ ID NO: 69; and 11 bp (GTTTCAGAACC;SEQ ID NO:159) deletion from position 3008 of SEQ ID NO: 69 giving rise to the genomic sequence of SEQ ID NO: 149. Out-of-frame mutation leading to an early stop codon and a protein with the amino acid sequence of SEQ ID NO: 150; Petition 870250103857, dated 11 / 13 / 2025, pages 175 / 212 166 / 166 Plant Name Phenotype Edition Description Comments CE197158 Spineless 5 bp deletion (AAGAC) starting at position 2922 of SEQ ID NO: 69; and 5 bp deletion (GCGCT) starting at position 2958 of SEQ ID NO: 69; resulting in the genomic sequence of SEQ ID NO: 151 Out-of-frame mutation leading to an early stop codon and a protein with the amino acid sequence of SEQ ID NO: 152 CE197159 Spineless 3 bp insertion (CGG) after position 2955 of SEQ ID NO: 69; and 1 bp deletion (T) at position 3010 of SEQ ID NO: 69; giving rise to the genomic sequence of SEQ ID NO: 153. Out-of-frame mutation leading to an early stop codon and a protein with the amino acid sequence of SEQ ID NO: 154. CE196211. No spikes. 8 bp deletion (CGGCGCTG) starting at position 2956 of SEQ ID NO: 69 giving rise to the genomic sequence of SEQ ID NO: 155. Out-of-frame mutation leading to an early stop codon and a protein with the amino acid sequence of SEQ ID NO: 156.

[00328] The foregoing is illustrative of the present invention and should not be construed as limiting it. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

1. A plant of Rosaceae or part thereof, characterized in that it comprises at least one mutation in at least one (e.g., one or more) functional endogenous WUSCHEL (WUS)-Homeobox related (WOX) gene encoding a WOX transcription factor.

2. A Rosaceae plant or part thereof, according to claim 1, characterized in that the endogenous functional gene WOX is either an endogenous functional gene WOX1 or an endogenous functional gene WOX23.

3. A Rosaceae plant or part thereof, according to claim 1 or 2, characterized in that the endogenous functional gene WOX is specifically expressed in tissues that give rise to trichomes, spines and / or prickles.

4. A Rosaceae plant or part thereof, according to any one of claims 1 to 3, characterized in that the endogenous functional gene WOX is specifically expressed in the vascular tissue, epidermal tissue and / or cells or tissues that give rise to thorns and / or prickles in the Rosaceae plant or part thereof.

5. A Rosaceae plant or part thereof, according to any of the preceding claims, characterized in that the WOX transcription factor regulates the production of thorns and / or prickles.

6. A plant of Rosaceae or part thereof, according to any of the preceding claims, characterized in that at least one mutation results in a dominant mutation, a semi-dominant mutation, or a hypomorphic mutation.

7. A Rosaceae plant or part thereof, according to any of the preceding claims, characterized by the fact that at least one mutation is a base substitution, a base deletion, a base insertion and / or an inversion.

8. A plant of Rosaceae or part thereof, according to any of the preceding claims, characterized in that at least one mutation comprises a base substitution at A, T, G, or C.

9. A plant of Rosaceae or part thereof, according to any of the preceding claims, characterized in that at least one mutation is a deletion of at least one base pair, optionally one base pair for about 100 consecutive base pairs.

10. A plant of Rosaceae or part thereof, according to any of the preceding claims, characterized in that at least one mutation is an insertion of at least one base pair.

11. A Rosaceae plant or part thereof, according to any one of claims 2 to 10, characterized in that at least one mutation is located in the first exon and / or second exon of the WOX1 gene and / or in the second exon of the WOX23 gene.

12. A Rosaceae plant or part thereof, according to any of the preceding claims, characterized in that at least one mutation is an out-of-frame insertion or an out-of-frame deletion.

13. A Rosaceae plant or part thereof, according to any of the preceding claims, characterized in that at least one mutation results in a mutation of the WOX gene that produces a truncated WOX transcription factor or does not produce any detectable WOX transcription factor.

14. A Rosaceae plant or part thereof, according to claim 12 or 13, characterized in that the out-of-frame insertion or out-of-frame deletion produces / results in a premature stop codon, optionally resulting in a C-terminal truncation of the encoded transcription factor (e.g., a truncated WOX transcription factor).

15. A Rosaceae plant or part thereof, according to claim 14, characterized in that the C-terminal truncation is a deletion of one amino acid residue for about 320 consecutive amino acid residues of the encoded WOX transcription factor.

16. A Rosaceae plant or part thereof, according to any of the preceding claims, characterized in that the Rosaceae plant or part thereof comprises at least one mutation in two or more endogenous functional WOX genes.

17. A Rosaceae plant or part thereof, according to any of the preceding claims, characterized in that the endogenous functional gene WOX: (a) comprises a nucleotide sequence with at least 80% sequence identity with any of the SEQ ID NOs: 69, 70, 75, 76, 128, 129 or 162-173; (b) comprises a region with at least 80% sequence identity with a nucleotide sequence of any of the following SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134; (c) encodes an amino acid sequence with at least 80% sequence identity to any of the following SEQ ID NOs: 71, 77, 130, or 174-182; and / or (d) encodes a region of an amino acid sequence with at least 80% sequence identity to any one of SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 136, or SEQ ID NO: 137.Petition 870250103857, dated 11 / 13 / 2025, p. 179 / 212 4 / 34.

18. A Rosaceae plant or part thereof, according to any of the preceding claims, characterized in that at least one mutation results in a deletion or insertion of one or more base pairs located in a region of the endogenous functional WOX gene, the region having at least 80% sequence identity with any of the nucleotide sequences of SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134.

19. A Rosaceae plant or part thereof, according to any of the preceding claims, characterized in that the Rosaceae plant comprising at least one mutation has a reduced number of thorns and / or prickles, thorns and / or prickles that have a modified architecture, thorns and / or prickles that are reduced in size and / or absence of thorns and / or prickles when compared to a control Rosaceae plant lacking at least one mutation.

20. A Rosaceae plant or part thereof, according to any of the preceding claims, characterized in that the Rosaceae plant or part thereof is a Rosa spp. plant or part thereof, a Hulthemia spp. plant or part thereof, a Hesperrhodos spp. plant or part thereof, a Platirhodon spp. plant or part thereof, or a Rubus spp. plant or part thereof.

21. A plant of Rosaceae or part thereof, according to claim 20, characterized in that the Rubus spp. are blackberry, black raspberry or red raspberry.

22. A Rosaceae plant or part thereof, according to any of the preceding claims, characterized in that the Rubus plant is Rubus allegheniensis (Allegheny blackberry), Rubus armeniacus (Himalayan blackberry), Rubus canadensis (Canadian blackberry), Rubus fruticosus agg.(European blackberry), Rubus laciniatus (perennial cut-leaf blackberry), Rubus pensilvanicus (Pennsylvanian blackberry), Rubus trifidus (Japanese blackberry), Rubus ursinus (wild blackberry), Rubus hybrid (e.g., progeny resulting from the hybridization of two or more of the preceding Rubus species), Rubus leucodermis (white-skinned raspberry or western black raspberry), Rubus occidentale (black raspberry), Rubus crataegifolius (Asian raspberry), Rubus gunnianus (Tasmanian alpine raspberry), Rubus idaeus (red raspberry or European red raspberry), Rubus parvifolius (Australian native raspberry), Rubus phoenicolasius (wine raspberry or blackberry), Rubus rosifolius (Mauritius raspberry), Rubus strigosus (American red raspberry) (syn. R. idaeus var. strigosus), or Rubus ellipticus (Himalayan yellow raspberry).

23. A Rosaceae plant or part thereof, according to any of the preceding claims, characterized in that at least one mutation results in a mutation of the WOX gene comprising a nucleotide sequence with at least 90% sequence identity with any of the SEQ ID NOs: 143, 144, 145, 147, 149, 151, 153 and / or 155 or encoding a WOX transcription factor with at least 90% sequence identity with any of the SEQ ID NOs: 146, 148, 150, 152, 154 and / or 156.

24. *A plant of Rosaceae or part thereof, according to any of the preceding claims, characterized in that the mutation is an unnatural mutation.

25. A Rosaceae plant cell, characterized in that it comprises an editing system, the editing system comprising: Petition 870250103857, 11 / 13 / 2025, p. 181 / 212 6 / 34 (a) a CRISPR-Cas effector protein; and (b) a guide nucleic acid (e.g., gRNA, gDNA, crRNA, crDNA, sgRNA, sgDNA) comprising a spacer sequence with complementarity to an endogenous functional target gene encoding a WUSCHEL (WUS)-related Homeobox (WOX) transcription factor in the Rosaceae plant cell.

26. A plant cell of Rosaceae, according to claim 25, characterized in that the WOX transcription factor is either a WOX1 transcription factor or a WOX23 transcription factor.

27. A plant cell of Rosaceae, according to claim 25 or 26, characterized in that the endogenous functional target gene is expressed in a tissue-specific manner, optionally in vascular tissue, epidermal tissue and / or cells or tissues that give rise to thorns and / or prickles.

28. A plant cell of Rosaceae, according to any one of claims 25 to 27, characterized in that the editing system generates a mutation in the endogenous functional target gene encoding the WOX transcription factor.

29. A plant cell of Rosaceae, according to any one of claims 25 to 28, characterized in that the endogenous functional target gene comprises two or more endogenous functional WOX genes.

30. A plant cell of Rosaceae, according to any one of claims 25 to 29, characterized in that the endogenous functional target gene: (a) comprises a nucleotide sequence with at least 80% sequence identity with any one of the SEQ ID NOs: 69, 70, 75, 76, 128, 129 or 162-173; (b) comprises a region with at least 80% sequence identity with a nucleotide sequence of any Petition 870250103857, dated 11 / 13 / 2025, p.182 / 212 7 / 34 one of the following SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134; (c) encodes an amino acid sequence with at least 80% sequence identity with any of the following SEQ ID NOs: 71, 77, 130, or 174-182; and / or (d) encodes a region of an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 136 or SEQ ID NO:

137.

31. Plant cell of Rosaceae, according to any one of claims 25 to 30, characterized in that the guide nucleic acid comprises any one of the nucleotide sequences of SEQ ID NOs: 138-142.

32. A Rosaceae plant, characterized in that it is regenerated from a part of the plant, as defined in any one of claims 1 to 24, or from a Rosaceae plant cell, as defined in any one of claims 25 to 31.

33. Rosaceae plant, according to claim 32, characterized in that the Rosaceae plant comprises a mutation of the WOX gene comprising a nucleotide sequence with at least 90% sequence identity with any of the SEQ ID NOs: 143, 144, 145, 147, 149, 151, 153 and / or 155 and / or encodes a WOX1 transcription factor with at least 90% sequence identity with any of the SEQ ID NOs: 146, 148, 150, 152, 154 and / or 156.

34. Rosaceae plant, according to claim 32 or 33, characterized in that the Rosaceae plant exhibits a phenotype of a reduced number of thorns and / or prickles, thorns and / or prickles that exhibit modified architecture, thorns and / or prickles that are reduced in size and / or absence of thorns and / or prickles when compared to a control Rosaceae plant devoid of at least one mutation.

35. A Rosaceae plant, according to any one of claims 32 to 34, characterized in that the WOX gene mutation comprises an unnatural mutation.

36. A plant cell of Rosaceae, characterized in that it comprises at least one mutation within an endogenous functional WOX gene, wherein at least one mutation is a base substitution, a base insertion, or a base deletion that is introduced using an editing system comprising a nucleic acid binding domain that binds to a target site within the endogenous functional WOX gene, wherein the endogenous functional WOX gene is specifically expressed in tissues that give rise to trichomes, spines, thorns, and / or prickles.

37. A plant cell of Rosaceae, according to claim 36, characterized in that the endogenous functional gene WOX is specifically expressed in the vascular tissue, epidermal tissue and / or cells or tissues that give rise to thorns and / or prickles in the Rosaceae plant or part thereof.

38. A plant cell of Rosaceae, according to claim 36 or 37, characterized in that the endogenous functional gene WOX is either an endogenous functional WOX1 gene or an endogenous functional WOX23 gene.

39. Rosaceae plant cell, according to any one of claims 36 to 38, characterized in that the endogenous functional gene WOX: (a) comprises a nucleotide sequence with at least 80% sequence identity with any one of the SEQ Petition 870250103857, dated 11 / 13 / 2025, p.184 / 212 9 / 34 ID NOs: 69, 70, 75, 76, 128, 129 or 162-173; (b) comprises a region with at least 80% sequence identity with a nucleotide sequence of any of the following SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134; (c) encodes an amino acid sequence with at least 80% sequence identity with any of the following SEQ ID NOs: 71, 77, 130, or 174-182; and / or (d) encodes a region of an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 126, 127, 136, or 137.

40. A plant cell of Rosaceae, according to any one of claims 36 to 39, characterized in that at least one mutation is a dominant mutation, a semi-dominant mutation, and / or a hypomorphic mutation.

41. A plant cell of Rosaceae, according to any one of claims 36 to 40, characterized in that the target site is located in the first exon and / or second exon of the WOX1 gene and / or in the second exon of the WOX23 gene.

42. A Rosaceae plant cell, according to any one of claims 36 to 41, characterized in that the target site is within a region of the endogenous WOX gene, the region having at least 80% sequence identity with any one of the SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134 and / or encoding an amino acid sequence having at least 80% sequence identity to any one of the SEQ ID NOs: 126, 127, 136 or Petition 870250103857, dated 11 / 13 / 2025, p. 185 / 212 10 / 34 137.

43. A Rosaceae plant cell, according to any one of claims 36 to 42, characterized in that the editing system further comprises a nuclease, the nucleic acid binding domain binds to a target site within a sequence with at least 80% sequence identity with any of the SEQ ID NOs: 69, 70, 75, 76, 128, 129, or 162-173 and / or in a sequence with at least 80% sequence identity with a sequence of any of the SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, and at least one mutation within a WOX gene is produced after cleavage by the nuclease.

44. A Rosaceae plant cell according to claim 43, characterized in that the nuclease is a zinc finger nuclease, transcription activator-like effector nucleases (TALENs), an endonuclease (e.g., Fok1), or a CRISPR-Cas effector protein.

45. A plant cell of Rosaceae, according to any one of claims 36 to 44, characterized in that the nucleic acid binding domain is a zinc finger, a transcription activator-like DNA binding domain (TAL), an argonaut, or a CRISPR-Cas effector nucleic acid binding domain.

46. ​​A plant cell of Rosaceae, according to any one of claims 36 to 45, characterized in that at least one mutation within the WOX gene is an insertion and / or a deletion, optionally at least one mutation is an out-of-frame insertion or an out-of-frame deletion.

47. A Rosaceae plant cell, according to any one of claims 36 to 46, characterized in that at least one mutation within the WOX gene is an insertion and / or a deletion that results in a premature stop codon, optionally resulting in a WOX transcription factor with a C-terminal truncation.

48. A plant cell of Rosaceae, according to claim 47, characterized in that the C-terminal truncation is the deletion of one amino acid residue for about 300 consecutive amino acid residues of the encoded WOX transcription factor.

49. A plant cell of Rosaceae, according to any one of claims 36 to 48, characterized in that at least one mutation within the WOX gene comprises a point mutation.

50. A plant cell of Rosaceae, according to any one of claims 36 to 49, characterized in that at least one mutation results in a WOX gene mutation comprising a nucleotide sequence with at least 90% sequence identity with any of the SEQ ID NOs: 143, 144, 145, 147, 149, 151, 153 and / or 155 and / or results in a WOX mutation gene encoding a WOX transcription factor with at least 90% sequence identity with any of the SEQ ID NOs: 146, 148, 150, 152, 154 and / or 156.

51. A plant cell of Rosaceae, according to any one of claims 36 to 50, characterized in that at least one mutation is an unnatural mutation.

52. A Rosaceae plant regenerated from a Rosaceae plant cell, according to any one of claims 36 to 51, characterized in that it comprises at least one mutation within the endogenous functional gene WOX.

53. Rosaceae plant, according to claim 52, characterized in that the Rosaceae plant comprising at least one mutation exhibits a reduced number of spines and / or prickles, spines and / or prickles having a modified architecture, spines and / or prickles that are reduced in size and / or no spines and / or prickles when compared to a control Rosaceae plant devoid of at least one mutation (e.g., an isogenic plant (e.g., unedited wild-type plant or a null segregant).

54. Method for producing / improving an edited and transgene-free Rosaceae plant, characterized in that it comprises: crossing the Rosaceae plant, according to any one of claims 1 to 24, 32 to 35, 52 or 53, with a transgene-free Rosaceae plant, thereby introducing at least one mutation into the Rosaceae plant that is transgene-free; and selecting a Rosaceae parent plant that comprises at least one mutation and is transgene-free, thereby producing an edited and transgene-free Rosaceae plant.

55. Method for providing a plurality of Rosaceae plants having a phenotype of a reduced number of thorns and / or prickles, thorns and / or prickles having a modified architecture, thorns and / or prickles that are reduced in size and / or no thorns and / or prickles, characterized in that it comprises planting two or more Rosaceae plants, according to any one of claims 1 to 24, 32 to 35, 52 or 53, in a cultivation area, thus providing a plurality of Rosaceae plants having a phenotype of a reduced number of thorns and / or prickles, thorns and / or prickles having a modified architecture, thorns and / or prickles that are reduced in size and / or no thorns and / or prickles compared with a plurality of control Rosaceae plants lacking at least one mutation.

56. Method for creating a mutation in a functional endogenous WUSCHEL (WUS)-Related Homeobox (WOX) gene in Petition 870250103857, dated 11 / 13 / 2025, p.188 / 212 13 / 34 a Rosaceae plant, characterized in that it comprises: (a) targeting a gene editing system to a portion of the endogenous functional WOX gene that (i) comprises a sequence with at least 80% sequence identity with any of the SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134; and / or (ii) encodes a sequence with at least 80% identity to any of the SEQ ID NOs: 126, 127, 136 or 137, and (b) select a Rosaceae plant comprising a modification located in a region of the endogenous functional WOX gene with at least 80% sequence identity to any of the SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134.

57. A method for generating variation in a WUSCHEL (WUS)-related Homeobox (WOX) transcription factor in a Rosaceae plant cell, characterized in that it comprises: introducing an editing system into a Rosaceae plant cell, wherein the editing system is directed to a region of a functional WUSCHEL (WUS)-related Homeobox (WOX) gene; and contacting the region of the functional WOX gene with the editing system, thereby introducing a mutation in the functional WOX gene and generating variation in the WOX transcription factor in the Rosaceae plant cell.

58. Method, according to claim 57, characterized in that the functional WOX gene is a functional WOX1 gene or a functional WOX23 gene.

59. Method according to claim 57 or 58, characterized in that the functional WOX gene comprises a nucleotide sequence with at least 80% sequence identity with SEQ ID NOs: 69, 70, 75, 76, 128, 129, or 162-173, and / or the region of the functional WOX gene in which the variation is generated comprises a nucleotide sequence with at least 80% sequence identity with any one of the SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125, or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134 and / or the WOX transcription factor comprises an amino acid sequence with at least 80% sequence identity with any of the SEQ ID Nos: 71, 77, 130, or 174-182, and / or the region of the WOX transcription factor in which the variation is generated comprises an amino acid sequence with at least 80% sequence identity with any of the SEQ ID Nos: 126, 127, 136 or 137.

60. A method according to any one of claims 57 to 59, characterized in that generating variation in the WOX transcription factor in a Rosaceae plant results in the Rosaceae plant having a reduced number of thorns and / or prickles, thorns and / or prickles that have a modified architecture, thorns and / or prickles that are reduced in size, and / or no thorns and / or prickles.

61. Method, according to any one of claims 57 to 60, characterized in that contacting the functional WOX gene region in the Rosaceae plant cell with the editing system produces a Rosaceae plant cell containing in its genome an edited endogenous WOX gene, the method further comprising (a) regenerating a Rosaceae plant from the plant cell. Petition 870250103857, dated 11 / 13 / 2025, p.190 / 212 15 / 34 ta of Rosaceae; (b) self-fertilize the Rosaceae plant to produce Rosaceae offspring plants (E1); (c) analyze the Rosaceae offspring plants from (b) for a reduced number of thorns and / or prickles, thorns and / or prickles that have a modified architecture, thorns and / or prickles that are reduced in size and / or absence of thorns and / or prickles; (d) select Rosaceae parent plants that have a reduced number of thorns and / or prickles, thorns and / or prickles that have a modified architecture, thorns and / or prickles that are reduced in size and / or no thorns and / or prickles in order to produce selected Rosaceae parent plants that have a reduced number of thorns and / or prickles, thorns and / or prickles that have a modified architecture, thorns and / or prickles that are reduced in size and / or no thorns and / or prickles compared to a control Rosaceae plant.

62. Method according to claim 61, characterized in that it further comprises (e) self-fertilization of selected descendant Rosaceae plants from (d) to produce descendant Rosaceae plants (E2); (f) testing the descendant Rosaceae plants from (e) for a reduced number of thorns and / or prickles, thorns and / or prickles having a modified architecture, thorns and / or prickles that are reduced in size and / or absence of thorns and / or prickles;(e) and (g) select Rosaceae parent plants that have a reduced number of thorns and / or prickles, thorns and / or prickles that have a modified architecture, thorns and / or prickles that are reduced in size and / or no thorns and / or prickles to produce selected Rosaceae parent plants that have a reduced number of thorns and / or prickles, thorns and / or prickles that have a modified architecture, thorns and / or prickles that are reduced in size and / or no thorns and / or prickles compared with a control Rosaceae plant, optionally repeating (e) to (g) one or more additional times.

63. A method for detecting a mutant WOX gene (a mutation in a functional endogenous WOX gene) in a Rosaceae plant is provided, characterized in that it comprises detecting in the Rosaceae plant genome a WOX gene that has at least one mutation within a region of the gene that has at least 80% sequence identity with a nucleotide sequence of any of the SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134.

64. Method according to claim 63, characterized in that the detected WOX mutant gene comprises a nucleotide sequence with at least 90% sequence identity with any of the SEQ ID NOs: 143, 144, 145, 147, 149, 151, 153 and / or 155 or encodes a polypeptide with at least 90% identity with any of the SEQ ID NOs: 146, 148, 150, 152, 154 and / or 156.

65. Method for editing a specific site in the genome of a Rosaceae plant cell, characterized in that it comprises: specific cleavage of a target site within an endogenous functional gene WUSCHEL (WUS)-related Homeobox (WOX) in the Rosaceae plant cell, the endogenous functional gene WOX: (a) comprising a nucleotide sequence with at least 80% sequence identity with any of the SEQ ID NOs: 69, 70, 75, 76, 128, 129 or 162-173; (b) comprising a region with at least 80% of Petition 870250103857, dated 11 / 13 / 2025, p.192 / 212 17 / 34 sequence identity with a nucleotide sequence of any of the SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134; (c) encode an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 71, 77, 130, or 174-182; and / or (d) encode a region of an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 126, 127, 136 or 137, thereby generating an edit in the functional endogenous WOX gene of the Rosaceae plant cell and producing a Rosaceae plant cell comprising the edit in the endogenous WOX gene.

66. Method according to claim 65, characterized in that the endogenous functional gene WOX is an endogenous functional gene WOX1 and / or an endogenous functional gene WOX23.

67. Method, according to claim 65 or 66, characterized in that the endogenous functional gene WOX is specifically expressed in tissues that give rise to trichomes, spines, thorns and / or prickles.

68. Method, according to any one of claims 65 to 67, characterized in that the endogenous functional WOX gene is specifically expressed in vascular tissue, epidermal tissue and / or cells or tissues that give rise to spines and / or prickles.

69. A method, according to any one of claims 65 to 68, characterized in that an edit is generated in two or more endogenous functional WOX genes.

70. Method, according to any one of claims 65 to 69, characterized in that it further comprises the regeneration of a Rosaceae plant from a Rosaceae plant cell comprising editing the endogenous functional gene WOX to produce a Rosaceae plant containing the edit in its endogenous functional gene WOX.

71. Method, according to any one of claims 65 to 70, characterized in that the edit is located in the first and / or second exon of the endogenous functional gene WOX.

72. A method, according to any one of claims 65 to 71, characterized in that the editing results in an unnatural mutation.

73. A method, according to any one of claims 65 to 72, characterized in that the Rosaceae plant comprising the edit in its endogenous functional WOX gene exhibits a phenotype of reduced number of spines and / or prickles, spines and / or prickles that have a modified architecture, spines and / or prickles that are reduced in size and / or without spines and / or prickles.

74. A method according to any one of claims 65 to 73, characterized in that the endogenous functional gene WOX encodes a WUSCHEL (WUS)-related homeobox (WOX) transcription factor and editing results in a truncated WOX transcription factor, optionally a C-terminal truncation of the WOX transcription factor, optionally wherein the C-terminal truncation results in the deletion of an amino acid residue for about 320 consecutive amino acid residues from the C-terminal of the WOX transcription factor.

75. Method, according to any one of claims 65 to 74, characterized in that the edit results in a mutation of the WOX gene comprising a nucleotide sequence with at least 90% sequence identity with any of the SEQ ID NOs: 143, 144, 145, 147, 149, 151, 153 and / or 155 or encodes a polypeptide with at least 90% identity with any of the SEQ ID NOs: 146, 148, 150, 152, 154 and / or 156.

76. Method for producing a Rosaceae plant, characterized in that it comprises: (a) contacting a population of Rosaceae plant cells comprising a functional endogenous WUSCHEL (WUS)-related Homeobox (WOX) gene with a nuclease linked to a nucleic acid binding domain (e.g., editing system) that binds to a sequence (i) having at least 80% sequence identity with a nucleotide sequence of any of the SEQ ID NOs: 69, 70, 75, 76, 128, 129, or 162-173, (ii) comprising a region having at least 80% identity with any of the SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125, or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134;(iii) encode an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 71, 77, 130, or 174-182, and / or (iv) encode a region with at least 80% sequence identity with any of the SEQ ID NOs: 126, 127, 136, or 137; (b) select a Rosaceae plant cell from the Rosaceae plant cell population in which an endogenous functional gene WOX has been mutated, thereby producing a Rosaceae plant cell comprising a mutation in the endogenous functional gene WOX; and (c) grow the selected Rosaceae plant cell into a Rosaceae plant comprising a mutation in the endogenous functional gene WOX.

77. Method for reducing the number and / or size of spines and / or prickles, to modify the architecture of spines and / or prickles. Petition 870250103857, dated 11 / 13 / 2025, p. 195 / 212 20 / 34 leos, and / or elimination of thorns and / or prickles in a Rosaceae plant, characterized in that it comprises (a) contacting a Rosaceae plant cell comprising an endogenous functional gene WUSCHEL (WUS) Homeobox related (WOX) with a nuclease targeting the endogenous functional gene WOX, wherein the nuclease is linked to a nucleic acid binding domain (e.g., editing system) that binds to a target site within the endogenous functional gene WOX, wherein the endogenous functional gene WOX: (i) comprises a nucleotide sequence with at least 80% sequence identity with any of the SEQ ID NOs: 69, 70, 75, 76, 128, 129 or 162-173;(ii) comprises a region with at least 80% sequence identity with a nucleotide sequence of any of the following: SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134; (iii) encodes an amino acid sequence with at least 80% sequence identity with any of the following SEQ ID NOs: 71, 77, 130, or 174-182; and / or (iv) encodes a region of an amino acid sequence with at least 80% sequence identity with any of the following SEQ ID NOs: 126, 127, 136 or 137 to produce a Rosaceae plant cell comprising a mutation in the endogenous functional gene WOX;and (b) the growth of a Rosaceae plant cell comprising a mutation in the endogenous functional gene WOX in a Rosaceae plant comprising the mutation in the endogenous functional gene WOX thus producing a Rosaceae plant with a Petition 870250103857, dated 11 / 13 / 2025, page 196 / 212 21 / 34 mutation in the endogenous gene WOX and reduced number of spines and / or prickles, spines and / or prickles with modified architecture, spines and / or prickles with reduced size and / or elimination of spines and / or prickles in the Rosaceae plant.; 78. Method for producing a Rosaceae plant or part thereof, characterized in that it comprises at least one cell with an endogenous mutation of the WUSCHEL (WUS) Homeobox related (WOX) gene, the method comprising contacting a target site within an endogenous functional WOX gene in the Rosaceae plant or part thereof with a nuclease comprising a cleavage domain and a nucleic acid binding domain, wherein the nucleic acid binding domain binds to a target site within the endogenous functional WOX gene, wherein the endogenous functional WOX gene: (a) comprises a nucleotide sequence with at least 80% sequence identity with any of the SEQ ID NOs: 69, 70, 75, 76, 128, 129 or 162-173;(b) comprises a region with at least 80% sequence identity with a nucleotide sequence of any of the following SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134; (c) encodes an amino acid sequence with at least 80% sequence identity with any of the following SEQ ID NOs: 71, 77, 130, or 174-182; and / or (d) encodes a region of an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 126, 127, 136 or 137, thereby producing the Rosaceae plant or part thereof comprising at least one cell with a mutation in the endogenous WOX gene. Petition 870250103857, dated 11 / 13 / 2025, p. 197 / 212 22 / 34; 79. Method for producing a Rosaceae plant or part thereof, characterized in that it comprises a mutated endogenous WUSCHEL (WUS)-related Homeobox (WOX) gene and exhibiting a reduced number of spines and / or prickles, spines and / or prickles exhibiting a modified architecture, spines and / or prickles that are reduced in size and / or without spines and / or prickles, the method comprising contacting a target site within an endogenous functional WOX gene in the Rosaceae plant or part thereof with a nuclease comprising a cleavage domain and a nucleic acid-binding domain, wherein the nucleic acid-binding domain binds to a target site within the endogenous functional WOX gene, wherein the endogenous functional WOX gene: (a) comprises a nucleotide sequence with at least 80% sequence identity with any of the SEQ ID NOs: 69, 70, 75, 76, 128, 129 or 162-173;(b) comprises a region with at least 80% sequence identity with a nucleotide sequence of any of the following SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134; (c) encodes an amino acid sequence with at least 80% sequence identity with any of the following SEQ ID NOs: 71, 77, 130, or 174-182; and / or (d) encodes a region of an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 126, 127, 136 or 137, thereby producing the Rosaceae plant or part thereof comprising the endogenous mutant gene WOX and exhibiting a reduced number of spines and / or prickles, spines and / or prickles that have a modified architecture, spines and / or prickles that are reduced in size and / or without spines and / or prickles.

80. Method, according to any one of claims 76 to 79, characterized in that the functional endogenous gene WOX is either an endogenous gene WOX1 or an endogenous gene WOX23.

81. A method, according to any one of claims 76 to 80, characterized in that the endogenous functional WOX gene is specifically expressed in tissues that give rise to trichomes, spines, thorns and / or prickles.

82. Method, according to any one of claims 76 to 81, characterized in that the endogenous functional WOX gene is specifically expressed in vascular tissue, epidermal tissue and / or cells or tissues that give rise to spines and / or prickles.

83. A method according to any one of claims 76 to 82, characterized in that the nuclease cleaves the endogenous functional WOX gene, thereby introducing the mutation into the endogenous functional WOX gene.

84. Method, according to any one of claims 76 to 83, characterized in that the mutation is an unnatural mutation.

85. Method, according to any one of claims 76 to 84, characterized in that the mutation is a substitution, an insertion, a deletion and / or an inversion.

86. Method, according to any one of claims 76 to 85, characterized in that the mutation is a dominant mutation, a semi-dominant mutation, or a hypomorphic mutation.

87. Method, according to any one of claims 76 to 86, characterized in that the mutation comprises a point mutation. Petition 870250103857, dated 11 / 13 / 2025, pp. 199 / 212 24 / 34 88. Method, according to any one of claims 76 to 87, characterized in that the mutation is an insertion of about one base pair to about 100 base pairs.

89. A method according to any one of claims 76 to 88, characterized in that the mutation is a deletion of one base pair to about 100 base pairs.

90. A method according to any one of claims 76 to 89, characterized in that the mutation is an out-of-frame insertion or an out-of-frame deletion, optionally resulting in a premature stop codon.

91. A method according to any one of claims 76 to 90, characterized in that the mutation results in a mutant WOX gene that produces a mutated WOX transcription factor, optionally wherein the mutated WOX transcription factor comprises a C-terminal truncation.

92. Method according to claim 91, characterized in that the mutated WOX transcription factor comprises a C-terminal truncation of an amino acid residue to about 325 consecutive amino acid residues, optionally a C-terminal truncation of about 200, 210, 220 consecutive amino acid residues to about 315 consecutive amino acid residues.

93. Method according to claim 91 or 92, characterized in that the mutant WOX gene comprises a nucleotide sequence with at least 90% sequence identity with any of the SEQ ID NOs: 143, 144, 145, 147, 149, 151, 153 and / or 155 or encodes a polypeptide with at least 90% identity with any of the SEQ ID NOs: 146, 148, 150, 152, 154 and / or 156.

94. Method, according to any of the claims Petition 870250103857, dated 11 / 13 / 2025, pp. 200 / 212 25 / 34 tions 76 to 93, characterized in that the nuclease is a zinc finger nuclease, transcription activator-like effector nucleases (TALEN), endonuclease or a CRISPR-Cas effector protein.

95. A method according to any one of claims 76 to 94, characterized in that the nucleic acid binding domain is a zinc finger, a transcription activator-like DNA binding domain (TAL), an argonaut, or a CRISPR-Cas effector DNA binding domain.

96. A method, according to any one of claims 76 to 95, characterized in that the Rosaceae plant or part thereof is a Rosa spp. plant or part thereof or a Rubus spp. plant or part thereof.

97. Method according to claim 96, characterized in that the Rubus spp. are blackberry, black raspberry or red raspberry.

98. Method, according to claim 96 or 97, characterized in that the Rubus plant is Rubus allegheniensis (Allegheny blackberry), Rubus armeniacus (Himalayan blackberry), Rubus canadensis (Canadian blackberry), Rubus fruticosus agg. (European blackberry), Rubus laciniatus (perennial cut-leaf blackberry), Rubus pensilvanicus (Pennsylvanian blackberry), Rubus trifidus (Japanese blackberry), Rubus ursinus (wild blackberry), Rubus hybrid (e.g., progeny resulting from the hybridization of two or more of the above Rubus species), Rubus leucodermis (white-skinned raspberry or western black raspberry), Rubus occidentale (black raspberry), Rubus crataegifolius (Asian raspberry), Rubus gunnianus (Tasmanian alpine raspberry), Rubus idaeus (red raspberry or European red raspberry), Rubus parvifolius (Australian native raspberry), Rubus phoenicolasius (wine raspberry or blackberry) Petition 870250103857, dated 11 / 13 / 2025, page.201 / 212 26 / 34 black), Rubus rosifolius (Mauritius raspberry), Rubus strigosus (American red raspberry) (syn. R. idaeus var. strigosus), or Rubus ellipticus (Himalayan yellow raspberry).

99. A Rosaceae plant, characterized in that it is produced by any of the methods defined in any of claims 76 to 98.

100. Guide nucleic acid that binds to a target site within a functional WUSCHEL (WUS)-related Homeobox (WOX) gene, characterized in that the target site is in a region of the functional WOX gene having at least 80% sequence identity with any of the SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134.

101. Guide nucleic acid, according to claim 100, characterized in that the guide nucleic acid comprises a spacer comprising a nucleotide sequence of any one of the following: SEQ ID NOS: 138-142.

102. System, characterized in that it comprises the guide nucleic acid, as defined in claim 100 or 101, and a CRISPR-Cas effector protein that associates with the guide nucleic acid.

103. System according to claim 102, characterized in that it further comprises a tracr nucleic acid that associates with the guide nucleic acid and a CRISPRCas effector protein, optionally wherein the tracr nucleic acid and the guide nucleic acid are covalently linked.

104. Gene editing system, characterized in that it comprises a CRISPR-Cas effector protein in association with a guide nucleic acid, wherein the guide nucleic acid comprises Petition 870250103857, dated 11 / 13 / 2025, page 202 / 212 27 / 34 of a spacer sequence that binds to a functional endogenous WUSCHEL (WUS)-related Homeobox (WOX) gene.

105. Gene editing system according to claim 104, characterized in that the endogenous functional gene WOX: (a) comprises a nucleotide sequence with at least 80% sequence identity with any of the SEQ ID NOs: 69, 70, 75, 76, 128, 129 or 162-173; (b) comprises a region with at least 80% sequence identity with a nucleotide sequence of any of the following SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134; (c) encodes an amino acid sequence with at least 80% sequence identity with any of the following SEQ ID NOs: 71, 77, 130, or 174-182; and / or (d) encodes a region of an amino acid sequence with at least 80% sequence identity with any of the SEQ ID NOs: 126, 127, 136, or 137.

106. Genetic editing system, according to claim 104 or 105, characterized in that the guide nucleic acid comprises a spacer sequence comprising a nucleotide sequence of any of the SEQ ID NOs: 138142.

107. Gene editing system, according to any one of claims 104 to 106, characterized in that it further comprises a tracr nucleic acid that associates with the guide nucleic acid and a CRISPR-Cas effector protein, optionally wherein the tracr nucleic acid and the guide nucleic acid are covalently linked. Petition 870250103857, dated 11 / 13 / 2025, pp. 203 / 212 28 / 34 108. Complex, characterized in that it comprises a guide nucleic acid and a CRISPR-Cas effector protein comprising a cleavage domain, wherein the guide nucleic acid binds to a target site within a functional endogenous WUSCHEL (WUS)-related Homeobox (WOX) gene, wherein the functional endogenous WOX gene: (a) comprises a nucleotide sequence with at least 80% sequence identity with any of the SEQ ID NOs: 69, 70, 75, 76, 128, 129 or 162-173; (b) comprises a region with at least 80% sequence identity with a nucleotide sequence of any of the following SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134; (c) encodes an amino acid sequence with at least 80% sequence identity with any of the following SEQ ID NOs: 71, 77, 130, or 174-182;and / or (d) encodes a region of an amino acid sequence with at least 80% sequence identity for any of the SEQ ID NOs: 126, 127, 136, or 137, and the cleavage domain cleaves a target strand in the WOX gene.

109. Expression cassette, characterized in that it comprises (a) a polynucleotide encoding the CRISPR-Cas effector protein comprising a cleavage domain and (b) a guide nucleic acid that binds to a target site within a functional endogenous WUSCHEL (WUS)-related Homeobox (WOX) gene, wherein the guide nucleic acid comprises a spacer sequence that is complementary to and binds to (i) a portion of a nucleic acid with at least 80% sequence identity with any of the SEQ ID Nos: 69, Petition 870250103857, dated 11 / 13 / 2025, p.204 / 212 29 / 34 70, 75, 76, 128, 129 or 162-173; (ii) a portion of a nucleic acid with at least 80% sequence identity with any of the SEQ ID NOs: 78-82, 83-86, 87-90, 91-109, 110-125 or 131-135, optionally SEQ ID NOs: 81, 82, 84-86, 88-90, 94-97, 99-101, 103-105, 107-109, 113-116, 118-120, 122, 124, 125 or 134; (iii) a portion of a nucleic acid that encodes an amino acid sequence with at least 80% sequence identity with any of the following SEQ ID NOs: 71, 77, 130, or 174-182; and / or (iv) a portion of a nucleic acid that encodes an amino acid sequence with at least 80% identity with any of the following SEQ ID NOs: 126, 127, 136, or 137.

110. Genetic marker for thorns and / or prickles with a modified architecture, a reduced number and / or size of thorns and / or prickles, or no thorns and / or prickles in a blackberry plant or part thereof, the genetic marker characterized in that it comprises (a) the nucleic acid sequence of GAACTCTG located between nucleotide positions 2897 and 2898 with reference to the nucleotide position numbering of SEQ ID NO: 69 and / or (b) the nucleic acid sequence of SEQ ID NO:

143.

111. Genetic marker, according to claim 110, characterized in that the blackberry plant is Rubus allegheniensis (Allegheny blackberry), Rubus armeniacus (Himalayan blackberry), Rubus canadensis (Canadian blackberry), Rubus fruticosus agg. (European blackberry), Rubus laciniatus (perennial cut-leaf blackberry), Rubus pensilvanicus (Pennsylvanian blackberry), Rubus trifidus (Japanese blackberry), Rubus ursinus (wild blackberry).

112. Method for identifying a blackberry plant or part thereof with a thorn and / or prickle phenotype with a Petition 870250103857, dated 11 / 13 / 2025, p.205 / 212 30 / 34 modified architecture, a reduced number and / or size of spines and / or prickles, or no spines and / or prickles, characterized in that it comprises the identification in the genome of a blackberry plant cell or part thereof of (a) an insertion in an endogenous functional WUSCHEL (WUS)-Homeobox related (WOX) gene, the insertion comprising the nucleic acid sequence GAACTCTG located between nucleotide positions 2897 and 2898 with reference to the nucleotide position numbering of SEQ ID NO: 69 and / or (b) the nucleic acid sequence of SEQ ID NO:

143.

113. Method for improving a blackberry plant with thorns and / or prickles with a modified architecture, a reduced number and / or size of thorns and / or prickles, or without thorns and / or prickles, comprising crossing a blackberry plant comprising in its genome (a) an insertion in an endogenous functional gene WUSCHEL (WUS)-Related Homeobox (WOX), the insertion characterized in that it comprises the nucleic acid sequence GAACTCTG located between nucleotide positions 2897 and 2898 with reference to the nucleotide position numbering of SEQ ID NO: 69 and / or (b) the nucleic acid sequence of SEQ ID NO: 143 with itself or with another blackberry plant to produce parent plants;and select one or more parent plants comprising the insertion and having thorns and / or prickles with a modified architecture, a reduced number and / or size of thorns and / or prickles, or no thorns and / or prickles, thus producing blackberry plants having thorns and / or prickles with a modified architecture, a reduced number and / or size of thorns and / or prickles, or no thorns and / or prickles.

114. Method, according to claim 113, characterized in that it further comprises crossing a selected parent plant with itself or with another blackberry plant one or more additional times and selecting parent plants comprising insertion, thereby producing blackberry plants with thorns and / or prickles with a modified architecture, with a reduced number and / or size of thorns and / or prickles, or without thorns and / or prickles.

115. Method, according to any one of claims 112 to 114, characterized in that the blackberry plant is Rubus allegheniensis (Allegheny blackberry), Rubus armeniacus (Himalayan blackberry), Rubus canadensis (Canadian blackberry), Rubus fruticosus agg. (European blackberry), Rubus laciniatus (perennial cut-leaf blackberry), Rubus pensilvanicus (Pennsylvanian blackberry), Rubus trifidus (Japanese blackberry), Rubus ursinus (wild blackberry).

116. Blackberry plant or part thereof, characterized in that it is produced by the method as defined in any one of claims 113 to 115, and exhibiting a thorn and / or prickle phenotype with a modified architecture, a reduced number and / or size of thorns and / or prickles, or no thorns and / or prickles compared to a control blackberry plant lacking the insertion.

117. Method for producing a Rosaceae plant, characterized in that it comprises a mutation in an endogenous WUSCHEL (WUS)-related Homeobox (WOX) gene and at least one polynucleotide of interest, the method comprising crossing a first Rosaceae plant, which is the Rosaceae plant as defined in any of claims 1 to 24, 32 to 35, 52, 53 or 99, with a second Rosaceae plant comprising at least one polynucleotide of interest to produce offspring Rosaceae plants; and Petition 870250103857, dated 11 / 13 / 2025, pp. 207 / 212 32 / 34 selecting Rosaceae progenitor plants comprising the mutation in the WOX gene and at least one polynucleotide of interest, thereby producing the Rosaceae plant comprising a mutation in an endogenous WOX gene and at least one polynucleotide of interest.

118. Method for producing a Rosaceae plant, characterized in that it comprises a mutation in an endogenous WOX gene and at least one polynucleotide of interest, the method comprising introducing at least one polynucleotide of interest into a Rosaceae plant as defined in any one of claims 1 to 24, 32 to 35, 52, 53 or 99, thereby producing a Rosaceae plant comprising a mutation in a WOX gene and at least one polynucleotide of interest.

119. Method according to claim 118 or 119, characterized in that the polynucleotide of interest is a polynucleotide that confers herbicide tolerance, insect resistance, disease resistance, increased yield, increased nutrient use efficiency, or resistance to abiotic stress.

120. Method for producing a Rosaceae plant, characterized in that it comprises a mutation in an endogenous WOX gene and exhibiting a phenotype of improved yield characteristics, improved plant architecture and / or improved defense characteristics, comprising crossing a first plant, which is the plant as defined in any of claims 1 to 24, 32 to 35, 52, 53 or 99, with a second plant exhibiting a phenotype of improved yield characteristics, improved plant architecture and / or improved defense characteristics; and selecting parent plants comprising the mutation. Petition 870250103857, dated 11 / 13 / 2025, p.208 / 212 33 / 34 mutation in the WOX gene and a phenotype of improved yield characteristics, improved plant architecture and / or improved defense characteristics, thus producing a plant comprising a mutation in an endogenous WOX gene and exhibiting a phenotype of improved yield characteristics, improved plant architecture and / or improved defense characteristics compared to a control plant.

121. A method for controlling weeds in a container (for example, a pot or seed tray and the like), a growing chamber, a greenhouse, a field, a recreation area, a lawn or a roadside, characterized in that it comprises applying a herbicide to one or more (a plurality) Rosaceae plants as defined in any one of claims 1 to 24, 32 to 35, 52, 53 or 99 grown in a container, a growing chamber, a greenhouse, a field, a recreation area, a lawn or a roadside, thereby controlling the weeds in the container, in the growing chamber, in the greenhouse, in the field, in the recreation area, on the lawn or on the roadside where one or more Rosaceae plants are growing.

122. Method for reducing insect predation on a Rosaceae plant, characterized in that it comprises applying an insecticide to one or more Rosaceae plants, as defined in any one of claims 1 to 24, 32 to 35, 52, 53 or 99, thereby reducing insect predation on one or more plants.

123. Method for reducing fungal diseases in a Rosaceae plant, characterized in that it comprises applying a fungicide to one or more Rosaceae plants, according to any of claims 1 to 24, 32-35, 52, 53 or 99, thus reducing the fungal disease in one or more Rosaceae plants.

124. A method according to claim 122 or 123, characterized in that one or more Rosaceae plants are growing in a container, a growth chamber, a greenhouse, a field, a recreation area, a lawn, or by the side of a road.

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