Solanum tuberosum with reduced polyphenol oxidase activity

Abstract

The invention relates to improvements in reduction of tuber browning in potato (Solanum tuberosum) varieties utilising endonuclease-directed gene editing technologies In particular, the invention provides a Solanum tuberosum plant cell comprising a mutation which generates a phenotype of reduced or eliminated polyphenol oxidase activity when compared to a Solanum tuberosum plant cell without the mutation. The invention further provides ribonucleoprotein complexes comprising an endonuclease and a guide RNA that hybridises with a target sequence comprised within a polyphenol oxidase 2 (StPPO2) gene of a Solanum tuberosum variety, and methods of using the same.

Description

[0001] SOLANUM TUBEROSUM WITH REDUCED POLYPHENOL OXIDASE ACTIVITY

[0002] FIELD OF THE INVENTION

[0003] The invention relates to improvements in reduction of tuber browning in potato (Solatium tuberosum) varieties utilising endonuclease-directed gene editing technologies.

[0004] BACKGROUND OF THE INVENTION

[0005] The potato (Solatium tuberosum) originated in Western South America where it grows wild in nature and presents the widest diversity of forms like tuber shape, size, colour, and flavour. The plant acquired great importance in the latter part of the sixteenth century C.E. when Spanish conquistadors brought it to Europe, after which is spread across the world along global trade routes. As a result, the potato has become the most cultivated tuber crop and the fourth most important global food crop, after wheat, rice and maize. It is a staple crop of many countries because it is easy to grow, requires less land than other major food crops and has high nutritional value. Improved agronomy and biotechnology efforts have resulted in a thousands of diverse cultivars and varieties that are able to generate improved yields as well as better adapt to climate stresses.

[0006] Potato belongs to family solanaceae and genus Solatium, with a basic set of 12 chromosomes. Potato is not only a widely used vegetable but also is used for making a wide range of processed foods. Potatoes are also used in industries for manufacturing starch, alcoholic beverages and even bio-based fuels. Development of varieties with agronomically important traits, and good keeping quality is one of the primary challenges for potato breeders.

[0007] Polyphenol oxidase (PPO), often encoded by a multi-gene family, causes oxidative browning, a significant problem in many food products and particularly in potato tubers. PPO is nearly ubiquitous in angiosperms and belongs to a class of copper-binding enzymes that catalyze the oxidation of phenolics to quinones. The subsequent non-enzymatic polymerization of the quinones leads to formation of brown pigments, sometimes called “black spot”, that are the cause of post-harvest deterioration (e.g. bruising) and loss of quality in stored potatoes. Losses caused by the browning resulting from PPO catalyzed-oxidations account for 50% of the losses of industrial production of fruits and vegetables. It was estimated that the total economic cost of potato bruising in the USA was USD 298.9 million in 1994 alone (Brook (1996), “Potato Bruising - How and Why Emphasising Black Spot Bruise”, Running Water Publishing, ISBN: 0-9650498- 0-9). Potato bruising may therefore have an economic impact in the USA in excess of USD 600 million in 2025. In addition to the undesired browning activity, the members of the PPO gene family appear to play important roles in signal transduction, stress and defence response throughout plant growth and development. Consequently, simple knock out of all the PPO genes is not a viable approach to generate improved potato varieties.

[0008] US-8889964-B1 describes a novel potato variety, called “W8”, that is transformed with nucleic acid sequences that are native to the potato plant genome. The DNA inserted into the genome of the potato variety W8 is a non-coding polynucleotide that silences genes involved in the phenotypes of black spot bruises, asparagine accumulation and senescence sweetening. The patent describes a plant vector, referred to as pSIM1278, that comprises a first silencing cassette containing two copies of a DNA segment comprising, in anti-sense orientation, a fragment of the asparagine synthetase-1 gene (fAsnl) and the 3'-untranslated sequence of the polyphenol oxidase-5 gene; and a second silencing cassette containing two copies of a DNA segment comprising, in anti-sense orientation, a fragment of the potato phosphorylase-L (pPhL) gene and a fragment of the potato R1 gene. In this way, the tubers of the potato plant variety expressing at least one silencing cassette display two or more desirable traits that are not present in the tubers of untransformed plants of the same variety.

[0009] WO-2018 / 035456-A1 describes a potato plant, plant part, or plant cell containing a deletion mutation in at least one PPO allele endogenous to the plant that leads to reduced PPO expression compared to a control potato plant. The mutation is made using a transcription activator-like effector endonuclease (TALE- nuclease) targeted to a POT32 allele or a POT33 allele.

[0010] Gonzalez et al. (Plant Cell Tiss Organ Cult 145, 291-305 (2021)) describes Agrobacterium tumefaciens-mediated transformation and protoplasts transfection delivery of CRISPR / Cas9 components directed to StPPO2 gene in the potato variety called Desiree. Two sgRNAs were designed to simultaneously direct Cas9 to the StPPO2 gene. The authors reported that the dual-sgRNA strategy resulted in poor performance due to low incidence of the targeted deletion. In addition, the reliance on a dual-sgRNA strategy increases the risk of un-intended modifications, such as off-target mutations and / or a large deletion in the target site. Furthermore, Gonzalez et al., sgRNAs were generated from DNA molecules as opposed to generated with chemical synthesis, which further increases their likelihood of creating unintended insertions in the genome.

[0011] Hence, there remains a need to provide a greater range of options for obtaining a non-browning phenotype in potatoes. There remains a need to create a novel set of allelic variants that can confer potato non-browning phenotype across a wide range of potato varieties. This is of particular importance because different specialised potato varieties are utilised for different purposes e.g. processed foods, potato chips (crisps), French fries, or table stock. These and other uses, features and advantages of the invention should be apparent to those skilled in the art from the teachings provided herein.

[0012] SUMMARY OF THE INVENTION

[0013] In a first aspect of the invention a Solanum tuberosum plant cell is provided comprising a mutation in at least one allele of a StPPO2 gene within a region 700-1200 base pairs from the ATG start codon of the StPPO2 gene, wherein the mutation generates a phenotype of reduced or eliminated polyphenol oxidase activity when compared to a Solanum tuberosum plant cell without the mutation.

[0014] A second aspect of the invention provides a Cas12a ribonucleoprotein (RNP) complex comprising a Cas12a endonuclease and a guide RNA (gRNA) that hybridises with a target sequence comprised within a polyphenol oxidase 2 (StPP02) gene of a Solanum tuberosum variety. In embodiments of the invention the gRNA hybridises with a target sequence comprised within a StPPO2 gene of more than one Solanum tuberosum variety, typically at least two, three or more than three varieties.

[0015] A third aspect of the invention provides a potato tuber comprising a plant cell of as described herein. Suitably, the potato tuber is resistant to PPO mediated browning reactions.

[0016] A fourth aspect of the invention provides a Solanum tuberosum plant or plant part thereof comprising a plant cell as described herein, wherein the plant exhibits a phenotype of reduced PPO mediated browning reactions compared to a non-genetically modified Solanum tuberosum plant.

[0017] A fifth aspect of the invention provides a tissue culture of regenerable cells of a plant or part thereof as described herein.

[0018] A sixth aspect of the invention provides an isolated guide RNA comprising a nucleotide sequence selected from any one of SEQ ID Nos: 2-4.

[0019] A seventh aspect provides a method for reducing or eliminating PPO activity in a plant cell from a Solanum tuberosum species the method comprising mutating one or more alleles of an StPPO2 gene within the genome of the plant cell, wherein the mutation is a frameshift mutation resulting from an insertion or deletion located within a region 700-1200 base pairs from an ATG start codon of the StPPO2 gene, and wherein the mutation is the result of a CRISPR / Cas gene editing event. Within the scope of this disclosure it is expressly intended that the various aspects, embodiments, examples and alternatives set out in the preceding paragraphs, in the claims and / or in the following description and drawings, and in particular the individual features thereof, may be taken independently or in any combination. That is, all embodiments and / or features of any embodiment can be combined in any way and / or combination, unless such features are incompatible.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021] One or more embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0022] Figure 1 is a representation of aligned consensus alleles from four potato varieties shown to determine conserved regions among varieties. This enables the choice of suitable exemplary sgRNAs based on perfect or near-perfect matches to the recognition sequence in each variety. The locations and 5”-3”orientations of a range of tested sgRNAs are shown. Vertical rectangular bars in sgRNA_34 and sgRNA_14 indicate single nucleotide polymorphisms (SNPs) in comparison to the Solanum tuberosum reference genome.

[0023] Figure 2 is a graph showing the protoplast transfection and StPPO2 gene editing efficiency (%GE) for the five sgRNAs identified in Figure 1 in a private potato variety designated “G”.

[0024] Figure 3 shows StPPO2 gene editing efficiency (%GE) in protoplasts using PEG transfection and Cas12a (ErCas12a) Ribonucleoprotein in four potato varieties using guide RNAs sgRNA_14 and sgRNA_28.

[0025] Figure 4 shows sequence alignments for the sgRNA_28 guide RNA versus a reference strand (SEQ ID NO: 5) representing the location in the wild type StPPO2 allele, together with post-editing modified alleles (SEQ ID Nos: 6-8) taken from an individual plant having deletions of 4, 10 and 8 nucleotides respectively.

[0026] Figure 5 is a photograph showing gene edited potato plants that were regenerated from tissue culture.

[0027] Figure 6 shows the results of screening the browning phenotype in potato tubers from original and improved varieties. Figure 6A is a photograph of original and improved Atlantic and Russt Burbank tubers grated and incubated for 14 hours. Figure 6B shows photographs from four post-grating incubation timepoints of original and improved Variety “G” tubers. Figure 6C is a photograph of original and improved Variety “G” tubers grated and incubated for 14 hours. Figure 7 is a graph showing average foliage weight (Figure 7A) and tuber weight (Figure 7B) of original (unedited) and improved (edited) potato lines.

[0028] Figure 8 shows the results of enzymatic assays of non-browning in original and improved potato lines. Figure 8A is a graph showing results of PPO enzymatic activity in original and improved Variety “G” lines. Figure 8B is a graph showing results of PPO enzymatic activity in original and improved Atlantic lines. Figure 8C is a graph showing results of PPO enzymatic activity in original and improved Russet Burbank lines.

[0029] Figure 9 is a graph showing results of enzymatic activity assay of StPPO2 in Variety “G” potato tubers from original lines and improved lines containing in-frame deletions of varying lengths.

[0030] Figure 10 is a graph depicting gene editing efficiency of sgRNA_28 (i.e. sg28) in different potato varieties.

[0031] DETAILED DESCRIPTION OF THE INVENTION

[0032] All references cited herein are incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Prior to setting forth the invention, a number of definitions are provided that will assist in the understanding of the invention.

[0033] Unless otherwise indicated, the practice of the present invention employs conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA technology, and chemical methods, which are within the capabilities of a person of ordinary skill in the art. Such techniques are also explained in the literature, for example, M.R. Green, J. Sambrook, 2012, Molecular Cloning: A Laboratory Manual, Fourth Edition, Books 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel, F. M. et al. (Current Protocols in Molecular Biology, John Wiley & Sons, Online ISSN:1934-3647); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D. McGee, 1990, In Situ Hybridisation: Principles and Practice, Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, IRL Press; and D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press; Synthetic Biology, Part A, Methods in Enzymology, Edited by Chris Voigt, Volume 497, pages 2-662 (2011); Synthetic Biology, Part B, Computer Aided Design and DNA Assembly, Methods in Enzymology, Edited by Christopher Voigt, Volume 498, Pages 2-500 (2011); RNA Interference, Methods in Enzymology, David R. Engelke, and John J. Rossi, Volume 392, Pages 1-454 (2005). All references cited herein are incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0034] As used herein, the term “comprising” means any of the recited elements are necessarily included and other elements may optionally be included as well. “Consisting essentially of’ means any recited elements are necessarily included, elements that would materially affect the basic and novel characteristics of the listed elements are excluded, and other elements may optionally be included. “Consisting of’ means that all elements other than those listed are excluded. Embodiments defined by each of these terms are within the scope of this invention.

[0035] A “polynucleotide” is a single or double stranded covalently-linked sequence of nucleotides in which the 3' and 5' ends on each nucleotide are joined by phosphodiester bonds. The polynucleotide may be made up of deoxyribonucleotide bases or ribonucleotide bases. Polynucleotides include DNA and RNA, and may be manufactured synthetically in vitro or isolated from natural sources. Sizes of polynucleotides are typically expressed as the number of base pairs (bp) for double stranded polynucleotides, or in the case of single stranded polynucleotides as the number of nucleotides (nt). One thousand bp or nt equal a kilobase (kb). Polynucleotides of less than around 40 nucleotides in length are typically called “oligonucleotides”.

[0036] The term “isolated”, when applied to a polynucleotide sequence, denotes that the sequence has been removed from its natural organism of origin and is, thus, free of extraneous or unwanted coding or regulatory sequences. Isolated oligonucleotides may be fully or partially of synthetic origin, that is, synthesised chemically as opposed to derived from natural sources.

[0037] The adjective “isolated,” when applied to a polypeptide or a ribonucleoprotein complex refers to a substantially purified composition, or in the case of a ribonucleoprotein complex, at least one component being a substantially purified component. In further respect to an isolated ribonucleoprotein complex, preferably all components are substantially purified.

[0038] As used herein, the terms 3' (‘3 prime’) and 5' (‘5 prime’) take their usual meanings in the art, i.e. to distinguish the ends or directionality within polynucleotide sequences. A polynucleotide has a 5' and a 3' end and polynucleotide sequences are conventionally written in a 5' to 3' direction. The 5’ end is suitably considered to be upstream of the 3’ end of a polynucleotide sequence. Hence, sequence referred to as upstream of a given reference point in a gene, such as the ATG transcription start codon of an open reading frame (ORF), is sequence that is 5’ to the reference point. Likewise sequence denoted as downstream is 3’ to the reference point, e.g. downstream to the ATG start codon. As used herein, the terms "plant" and "plant part" refer to cells, tissues, organs, seeds, and severed parts (e.g., roots, leaves, and flowers) that retain the distinguishing characteristics of the parent plant. "Tuber" refers to a thickened underground part of a stem or rhizome, serving as a food reserve and bearing buds from which new plants arise. "Seed" refers to any plant structure that is formed by continued differentiation of the ovule of the plant, following its normal maturation point at flower opening, irrespective of whether it is formed in the presence or absence of fertilization and irrespective of whether or not the seed structure is fertile or infertile. Other propagatable parts of a plant may include tissue cultures of regenerable cells, calli, cuttings, or root segments. Suitably, a “plant cell” may be selected from a gametophyte, a reproductive cell, a vegetative cell and / or a meristematic cell. In embodiments, suitable plant tissue is selected from: leaf, stem, root, tuber, seed, branch, pubescence, nodule, leaf axil, flower, pollen, stamen, pistil, petal, peduncle, stalk, stigma, style, bract, fruit, trunk, carpel, sepal, anther, ovule, pedicel, needle, cone, rhizome, stolon, shoot, pericarp, endosperm, placenta, berry, stamen, or leaf sheath. In specific embodiments plant cell material may include root tissue, leaf mesophyll and / or cultured callus.

[0039] In certain embodiments the plant cell is in the form of a protoplast. As used herein, the term “plant protoplasts” (also referred to simply as “protoplast”, throughout this disclosure) refers to a plant cell that has had its cell wall completely or partially removed. Removal of cell wall can be effected by mechanical, chemical or enzymatic means. In embodiments, protoplasts are obtained from suitable plant material using cell wall digestive enzymes. For example, enzymes such as cellulase, macerozyme, pectinase, hemicellulase, pectolyase, driselase, xylanase and combinations thereof may be suitable for use in the context of the invention. In embodiments, cellulase may be used at a concentration of 1w% - 1.5w%. In embodiments, macerozyme may be used at a concentration of 0.2w% - 0.4w%. In embodiments, hemicellulase may be used at a concentration of 2w% - 5w%. In embodiments, pectolyase may be used at a concentration 0.01w% - 0.5w%. In embodiments, driselase may be used at a concentration of 0.5w% - 2w%. Protocols for obtaining protoplasts from plant tissues are known in the art, for example in Yoo, Cho, & Sheen (2007) Nature Protocols volume 2, pages 1565-1572.

[0040] The term "allele(s)" means any of one or more alternative forms of a gene at a particular locus. In a diploid (or amphidiploid) cell of an organism, alleles of a given gene are located at a specific location or locus on a chromosome, with one allele being present on each chromosome of the pair of homologous chromosomes. Similarly, in a tetrapioid cell of an organism, one allele is present on each chromosome of the group of four homologous chromosomes. "Heterozygous" alleles are different alleles residing at a specific locus, positioned individually on corresponding homologous chromosomes. "Homozygous" alleles are identical alleles residing at a specific locus, positioned individually on corresponding homologous chromosomes in the cell. "Wild type" as used herein refers to a typical form of a plant or a gene as it most commonly occurs in nature.

[0041] The term “allelic variant” is used herein to denote any two or more alternative forms of a gene occupying the same chromosomal locus and controlling the same inherited characteristic. Allelic variation arises naturally though mutation, and may result in phenotypic polymorphism within populations. Gene mutations typically result in an altered nucleic acid sequence and in some cases an altered polypeptide sequence also. As used herein, the term “allelic variant” is additionally used to refer to the protein or polypeptide encoded by the allelic variant of a gene.

[0042] A "wild type PPO2 allele" is a naturally occurring PPO2 allele (e.g., as found within naturally occurring S. tuberosum plants, gene number PGSC0003DMG400018916) that encodes a functional PPO2 protein, while a "non-functional mutant PPO2 allele" is a PPO2 allele that does not encode a functional PPO2 protein. Such a "non-functional mutant PPO2 allele" can include one or more mutations in its nucleic acid sequence, where the mutation(s) result in reduced or even no detectable amount of functional PPO2 protein in the plant or plant cell in vivo or in extracts taken from mutant plant cells and tested in vitro.

[0043] The terms “eliminating”, “suppressing” or “reducing” when used in reference to a gene(s), refers to a lowering, reduction, or elimination of the expression level of a mRNA and / or protein product encoded by the gene(s), and / or a lowering, reduction, or elimination of the activity of a protein encoded by the gene(s) in a plant, plant cell or plant tissue, at one or more stage(s) of plant development, as compared to the expression level of such target mRNA and / or protein, and / or the activity of such encoded protein in a wild-type or control plant, cell or tissue at the same stage(s) of plant development. Lowering, reduction, or elimination may be determined functionally such as by testing the activity of the gene product, or via various quantitative measures such as those that assess reduction in production of protein product (e.g. Western blot, mass spectrometry, ELISA).

[0044] The genetically engineered or genetically edited potato cells or protoplasts described herein can be prepared using conventional gene editing methods or those described more specifically herein to edit one or more of target PPG genes. Targeted editing can be achieved either through a nuclease-independent approach, or through a nuclease-dependent approach. In the nuclease-independent targeted editing approach, homologous recombination is guided by homologous sequences flanking an exogenous polynucleotide to be introduced into an endogenous sequence through the enzymatic machinery of the host cell. The exogenous polynucleotide may introduce deletions, insertions or replacement of nucleotides in the endogenous sequence, such as within the identified region of the StPPO2 gene. The mutations to the StPPO2 gene may serve to knock out the gene function entirely and / or may result in an allelic variant that has reduced or impaired PPO activity. Alternatively, a nuclease-dependent approach can achieve targeted editing with higher frequency through the specific introduction of double strand breaks (DSBs) by specific rare- cutting nucleases (e.g., endonucleases). Nuclease-dependent targeted editing of this type also utilizes host DNA repair mechanisms, for example, non-homologous end joining (NHEJ), which occurs in response to these DSBs. DNA repair by NHEJ can lead to random insertions or deletions (so called “indels”) of a small number of endogenous nucleotides at the cleavage site. In contrast to NHEJ mediated repair, repair can also occur by a homology directed repair (HDR). When a donor template containing exogenous genetic material flanked by a pair of homology arms is present, the exogenous genetic material can be introduced into the genome by HDR, which results in targeted integration of the exogenous genetic material.

[0045] In some embodiments, gene disruption may occur by deletion of a genomic sequence using two guide RNAs. Methods of using CRISPR-Cas gene editing technology to create a genomic deletion in a cell, such as to knock out a gene in a plant cell, are described in Nekrasov et al. Sci Rep 7, 482 (2017).

[0046] Available endonucleases capable of introducing specific and targeted DSBs include, but not limited to, zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and RNA-guided CRISPR-Cas nucleases (CRISPR-Cas; Clustered Regular Interspaced Short Palindromic Repeats Associated). Additionally, DICE (dual integrase cassette exchange) system utilizing phiC31 and Bxb1 integrases may also be used for targeted integration.

[0047] CRISPR-Cas systems are widespread prokaryotic adaptive immune systems that are best known as components of a next generation of genome-editing tools. Cas9 recognizes 3' G-rich PAMs; it has become the most widely used CRISPR-Cas system and has been adapted for genome editing in various contexts, including in plants. Unlike Cas9, Cas12a recognizes 5' T- rich PAMs and self-processes its CRISPR RNA (crRNA). According to certain embodiments of the present invention an RNA-guided CRISPR-Cas12a nuclease (CRISPR / Cas12a) nuclease is utilised, suitably a ErCas12a (MAD7) (US patent number US9982279B1) (https: / / www.inscripta.com / madzymes / faq / ). MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas (Cas12a / Cpf1) family with a low level of homology to canonical Cas12a nucleases, that is particularly effective at generating indel mutations in plant cells. Other Nonlimiting examples of RNA-guided endonuclease systems include Cas1 , Cas1 B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Casio, Csy1 , Csy2, Csy3, Cse1 , Cse2, Csc1 , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1 , Cmr3, Cmr4, Cmr5, Cmr6, Csb1 , Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1 , Csx15, Csf1 , Csf2, Csf3, and Csf4, as well as homologs thereof, or modified versions thereof. CRISPR-Cas gene editing technology involves the use of a genome-targeting nucleic acid that can direct the endonuclease to a specific target sequence within a target gene for gene editing at the specific target sequence. The genome-targeting nucleic acid can be an RNA. A genometargeting RNA is referred to as a “guide RNA” or “gRNA” herein. A guide RNA typically comprises at least a spacer (or protospacer) sequence that hybridizes to a target nucleic acid sequence within a target gene for editing, and a CRISPR repeat sequence (the crRNA). In Type V gRNAs, of the type that is utilised by Cas12a nucleases (e.g. ErCas12a / MAD7), the crRNA forms a duplex that binds to the endonuclease, such that the guide RNA and endonuclease form a complex. In some embodiments, the genome-targeting nucleic acid provides target specificity to the complex by virtue of its association with the endonuclease. The genometargeting nucleic acid thus directs the activity of the endonuclease to a specific target site within the genome of a host cell. Hence, it will be understood by the person of ordinary skill in the art, that each gRNA is designed to include a spacer sequence complementary to its genomic target sequence. The term guide RNA (gRNA) is used synonymously with single guide RNA, “sgRNA”.

[0048] In embodiments of the present invention, the spacer sequence comprised within the gRNA is an oligonucleotide sequence, typically around 20 to 25 nucleotides in length, that defines a target sequence (e.g., a DNA target sequence, such as a genomic target sequence) of a target gene of interest. In embodiments of the present invention the target is a region within the StPPO2 gene. In some embodiments, the spacer sequence ranges from 15 to 30 nucleotides in length. For example, the spacer sequence may contain at least 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, a spacer sequence contains at least 22 nucleotides.

[0049] In embodiments of the present invention, the selected “target sequence” is in a target gene, suitably a StPPO2 gene, that is adjacent to a PAM sequence and is the sequence to be modified by an RNA-guided endonuclease (e.g., Cas12a, or MAD7). In specific embodiments of the present invention the region of the StPPO2 gene identified as having a suitable number of potential target sequences is between 700-1200 base pairs from the start codon (ATG) of the StPPO2 gene. In specific embodiments of the present invention the target sequence is in the region of the StPPO2 gene 700-800 base pairs from the start codon (ATG) of the StPPO2 gene. In specific embodiments of the present invention the target sequence is in the region of the StPPO2 gene 1100-1200 base pairs from the start codon (ATG) of the StPPO2 gene. In specific embodiments of the present invention the target sequence is comprised of two target sequences in the region of the StPPO2 gene 700-800 and 1100-1200 base pairs from the start codon (ATG) of the StPPO2 gene. The “target sequence” is on the so-called PAM-strand in a “target nucleic acid,” which is a double-stranded DNA molecule containing the PAM-strand and a complementary non-PAM strand. One of skill in the art recognizes that the gRNA spacer sequence hybridizes to the complementary sequence located in the non-PAM strand of the target nucleic acid of interest. The spacer sequence of a gRNA interacts with a target nucleic acid of interest in a sequence-specific manner via Watson-Crick base pairing. The nucleotide sequence of the spacer thus varies depending on the target sequence of the target nucleic acid of interest.

[0050] The gRNA disclosed herein may target any sequence of interest via the spacer sequence comprised within it. In some embodiments, the amount of complementarity between the spacer sequence of the guide RNA and the target sequence in the target gene can be about 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the spacer sequence of the guide RNA and the target sequence in the target gene is 100% complementary. In other embodiments, the spacer sequence of the guide RNA and the target sequence in the target gene may contain up to 5 mismatches, e.g., up to 4, up to 3, up to 2, or up to 1 mismatch. Typically, the mismatches are not consecutive and may be distributed across the spacer sequence. In embodiments of the present invention, a single gRNA (sgRNA) is employed as part of the RNP complex of the invention.

[0051] Any of the gRNAs disclosed herein may be chemically unmodified - i.e. utilising naturally occurring nucleotides. Alternatively, the gRNAs may contain one or more modified nucleotides and / or modified backbones. For example, a modified gRNA can comprise one or more 2'-O- methyl phosphorothioate nucleotides, which may be located at either the 5' end, the 3' end, or both. Alternatively, the gRNAs may comprise at least one non-naturally occurring nucleotide such as any one of those described in US-2020 / 0224234-A1 .

[0052] Exemplary spacer sequences suitable for inclusion in gRNAs targeting an StPPO2 gene are provided in Table 2 below. In embodiments of the invention the spacer sequences hybridize with an antisense strand of the stPPO2 gene at a location around 700 to around 1200 nucleotides downstream of the start codon (ATG). In further embodiments, spacer sequences hybridize with an antisense strand of the stPPO2 gene at a location around 700 to around 800 or at a location around 1100 to around 1200 nucleotides downstream of the start codon (ATG). The exemplary spacer sequences are particularly suitable for inclusion within a gRNA intended for use with a Cas12a based gene editing system, most typically with ErCas12a / MAD7. The exemplary spacer sequences show unexpectedly good utility in effecting a gene editing event resulting in a mutation in the genome of more than one distinct variety of Solanum tuberosum. Hence, unlike previous gene editing strategies the gRNAs provided herein can be used in multiple heterogenous varieties to reduce or eliminate undesirable PPO2 mediated browning reactions in potato tubers. The gRNAs provided herein may achieve robust, high editing efficiency across varieties whilst introducing few or no off-target effects or off-target edits into the Solanum tuberosum genome.

[0053] Cas12a RNA guided endonucleases, such as ErCas12a / MAD7, will bind to genomic regions matching the designed RNA spacer sequence that are adjacent to a suitable immediate upstream to a PAM site. In this location, the MAD7 endonuclease enzyme uses a single RuvC-like endonuclease domain to cut the DNA in a staggered manner, leaving 4-nucleotide 5'-overhangs at the PAM distal end of the genomic target site. The two resulting cleavage sites are located 19 bases after the PAM on the PAM-strand (sense strand) and after 23 bases on the non-PAM strand (antisense strand). In contrast to Cas 12a endonucleases, such as MAD7, the canonical Streptococcus pyogenes Cas9 protein uses two nuclease domains (HNH and RuvC-like) to break each DNA strand and generate a nearly synchronous blunt-ended DSB proximal to the PAM. The ability of Cas12a RNA guided endonucleases to produce sticky ends upon cleavage of the target DNA enhances the generation of indels, thus, facilitating knock out type frame-shift mutations at the target site. The mutations to the StPPO2 gene may serve to knock out the gene function entirely or may result in an allelic variant that has reduced or impaired PPO2 activity.

[0054] The site-directed endonuclease systems disclosed herein, comprising one or more gRNAs and at least one RNA-guided nuclease, such as Cas12a, can be delivered to a target cell or protoplast (e.g., a potato cell or protoplast) for genetic editing of the target gene, via any conventional method. In some embodiments, components of an endonuclease system as disclosed herein may be delivered to a target cell separately, either simultaneously or sequentially. In other embodiments, the components of the endonuclease system may be delivered into a target together, for example, as a complex. In some instances, gRNA and the RNA-guided endonuclease can be pre-complexed together to form a ribonucleoprotein (RNP), which can be delivered into a target cell or protoplast using conventional techniques. Hence, as used herein the phrase “ribonucleoprotein complex” or “RNP” refers to a ribonucleoprotein complex having CRISPR-associated endonuclease activity.

[0055] In embodiments of the present invention, the potato (S. tuberosum) plants, cells, plant parts, seeds, other propagatable material and progeny thereof that are provided herein can have a mutation in the endogenous alleles of one or more StPPO2 genes, such that expression of a functional StPPO2 protein is reduced or completely inhibited. Thus, in some cases, the plants, cells, plant parts, seeds, other propagatable material and progeny exhibit substantially reduced or even entirely eliminated detectable levels of polyphenol oxidase activity. In specific embodiments of the invention the level of polyphenol oxidase activity in the plants, cells, plant parts, seeds, other propagatable material and progeny thereof is sufficiently reduced to substantially eliminate PPO catalysed browning reactions in tubers. In other embodiments of the invention the level of polyphenol oxidase activity in the plants, cells, plant parts, seeds, other propagatable material and progeny thereof is sufficiently reduced to substantially reduce PPO catalysed browning reactions (such as caused by accumulation of quinones) in tubers by at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 40%, 30% and 20% when compared to a tuber from the same variety that has not been subjected to a mutation within a StPPO2 gene. Potato bruising is an industry-wide concern which results in the wastage of large quantities of otherwise quality potato. Potato bruising occurs through the same enzymatic process as potato browning and is mediated by PPO. The total economic cost of potato bruising in the USA was USD 298.9 million in 1994 alone (Brook, 1996). In contemporary figures, potato bruising may therefore have an economic impact of USD 600 million in the USA alone. Often, potatoes may be stored for approximately a year after harvesting and often incur pressure bruising from storage conditions and from handling during harvesting and shipping. Bruised potatoes are often discarded resulting in a reduction in the marketable yield of the crop. Potato (S. tuberosum) plants, cells, plant parts, seeds, other propagatable material and progeny thereof according to the present invention may exhibit substantially reduced or even entirely eliminated detectable levels of polyphenol oxidase activity. As a result, the plants, cells, plant parts, seeds, other propagatable material and progeny described herein may have a reduced susceptibility to bruising, resulting in improved marketable yields.

[0056] According to embodiments of the invention, methods are provided for efficiently engineering and recovering plants, plant cells, or other propagatable plant material - e.g. seeds, cuttings, calli, protoplasts or any other tissue culture of regenerable cells - comprising a non-wild type PPO gene expression pattern, particularly non-wild type StPPO2 gene expression. Typically, the plants, plant cells, or other propagatable plant material are from Solanum species, more suitably varieties of Solanum tuberosum - i.e. potato varieties, of which several thousand exist. There are two major subspecies of potato (Solanum tuberosum)-, andigena, or Andean; and tuberosum, or Chilean. In general, well-known cultivated varieties include, but are not limited to, russets, reds, whites, yellows (also called Yukons) and purples. Popular varieties, also known as cultivars, include, but are not limited to, those selected from: Abbot, Accent, Adirondack Blue, Adirondack Red, Agata, Agria, Almond, Alturas, Amandine, Americar, Amin, Annabelle, Anya, Arran Victory, Atlantic, Arizona, Austrian Crescent, Bamberg, Belana, Belle de Fontenay, BF-15, Bildtstar, Bintje, Blackberry, Blue Congo, Blue Danube, Bonnotte, Canela Russet, Cara, Caribou Russett, Cabritas, Camota, Cardinal, Centennial Russet, Challanger, Charlotte, Chelina, Chieftain, Chiloe, Cielo, Ciklamen, Clavela Blanca, Clearwater Russet, Colleen, Congo, Cosmos, Dakota Russett, Desiree, Draga, Duke of York, Edzell Blue, Elba, Faraja, Feldeslohn, Fianna, Fingerling, Flava, Fontana, Frisia, Gala, Georgina, German Butterball, Gold Rush, Golden Wonder, Golof, Hannah Sweet, Hunter, Innovator, Japanese Sweet, Jersey Royal, Kerr”s Pink, Kennebec, Kenya Baraka, Kestrel, King Edward, Kinongo, Kipfler, Kuras, Lady Balfour, Lady Crystal, Lady Felicia, Lady Rosetta, Lamoka, Laura, Linda, Magic Molly, Marfona, Marilynn, Maris Bard, Maris Piper, Maris Peer, Marquis, Mellody, Meru Mix, Moonlight, Mozart, Mukorino, Nadine, Natascha, Nectar, Ngure, Nicola, Norland, Novello, Nyayo Mukori, Orla, Pachacona, Patrones, Pimpernel, Pink Eye, Pink Fir Apple, Pinto Gold, Pirol, Primura, Ratte, Picasso, Purple Majesty, Purple Viking, Purplu, Ranger Russet, Red Gold, Red Norland, Red Pontiac, Red Rascal, Red Thumb, Reveille Russet, Robijn, Robinta, Rockstar, Romano, Rooster, Roslin Chania, Roslin Tana, Roslin, Royal, Rudolph, Rua, Rugano, Russet Burbank (selections), Russet Norkotah (selections), Sante, Sarpo Mira, Sasamua Satina, Selma, Setanta, Shepody, Sieglinde, Sirco, Soraya, Spunta, Snowden, Stobrawa, Strawberry Paw, Umatilla Russet, Urgentia, Valor, Variety “G”, Vivaldi, Vitelotte, Waneta, Wilja, Yellow Finn, and Yukon Gold. Any or substantially all cultivated varieties may be transformed or modified as disclosed herein. In particular embodiments of the invention the potato varieties are selected from Russet Burbank and / or Russet Norkotah 278.

[0057] The inventors have found that StPPO2, despite being a relatively small gene, has a large number of PAM sites recognised by Cas12a enzymes, such as ErCas12a / MAD7 - i.e. a YTN, YTTN or YTTV site, where Y = C or T, N = any nucleobase and V = any base other than T - and therefore provides a promising range of possible target sites for gene editing. However, since StPPO2 belongs to the wider PPG gene family, many of the potential target sites are conserved between PPO genes and would likely result in undesirable off-target genome editing of homologous PPO genes in the potato genome. Furthermore, since potato varieties have highly polymorphic alleles, most of the target sites identified had one or several polymorphism events over the potential gRNA binding sequence for the DNA recognition site across multiple potato varieties, which could result in non-editing of the alleles with a different sequence. This was confirmed when trialling promising candidate sgRNAs across varieties. Hence, identification of promising sgRNAs that can be used across several varieties requires more than simple trial and error screening. In embodiments of the present invention, sgRNAs are provided that demonstrate high specificity and gene editing efficiency to the StPPO2 in a diverse range of potato varieties but which exhibit low or non-existent off target effects.

[0058] In specific embodiments of the present invention, there is provided a potato plant cell, tuber, and / or whole plant that comprises an insertion or deletion mutation in a region 700-1200 base pairs from the ATG start codon of at least one allele of the StPPO2 gene. In specific embodiments of the present invention, there is provided a potato plant cell, tuber, and / or whole plant that comprises an insertion or deletion mutation in a region 700-800 base pairs from the ATG start codon of at least one allele of the StPPO2 gene. In specific embodiments of the present invention, there is provided a potato plant cell, tuber, and / or whole plant that comprises an insertion or deletion mutation in a region 1100-1200 base pairs from the ATG start codon of at least one allele of the StPPO2 gene. In a specific embodiments of the present invention, there is provided a potato plant cell, tuber, and / or whole plant that comprises an insertion or deletion mutation in a region 700-800 base pairs and in a region 1100-1200 base pairs from the ATG start codon of at least one allele of the StPPO2 gene. The mutated potato plant cell, tuber, and / or whole plants described may be referred to as gene edited and / or modified. Suitably, the mutation may be within multiple alleles, or within all alleles present in the genome of the potato plant cell, tuber, and / or whole plant. In one embodiment of the invention, the mutation is a frameshift mutation suitably the result of an insertion or deletion during NHEJ or HDR repair of a staggered double strand break in the DNA. Optionally, the staggered double strand break in the DNA is due to a gene editing event as described herein. In one embodiment of the invention, the mutation is a deletion of less than around 20 nucleotides, less than around 15 nucleotides, less than around 10 nucleotides, and optionally less than around 5 nucleotides.

[0059] In some embodiments, the present invention provides a potato plant cell, tuber, and / or whole plant having an StPPO2 gene that has been modified. In some embodiments, the present invention provides a potato plant cell, tuber, and / or whole plant having an StPPO2 gene having a sequence comprising SEQ ID NO: 6, SEQ ID NO: 7, and / or SEQ ID NO: 8. In some embodiments, the present invention provides a potato plant cell, tuber, and / or whole plant having an StPPO2 gene having one or more alleles comprising SEQ ID NO: 6, SEQ ID NO: 7, and / or SEQ ID NO: 8.

[0060] In some embodiments, the present invention provides a potato plant cell, tuber, and / or whole plant comprising a mutation in at least one allele of a polyphenol oxidase 2 (StPPO2) gene within a region 700-1200 base pairs from the ATG start codon of the StPPO2 gene, wherein the mutation generates a phenotype of reduced or eliminated polyphenol oxidase activity when compared to a Solanum tuberosum plant cell without the mutation. In some embodiments, the mutation is within a region 700-800 base pairs from the ATG start codon of the StPPO2 gene. In some embodiments, the mutation is within a region 1100-1200 base pairs from the ATG start codon of the StPPO2 gene. In some embodiments, the potato plant cell, tuber, and / or whole plant comprises a mutation in at least two alleles within the StPPO2 gene, wherein the mutations are within a region 700-800 base pairs and 1100-1200 base pairs from the ATG start codon of the StPPO2 gene.

[0061] In further embodiments, the present invention provides for uses of plants, plant parts, cells or seed of as described herein in agriculture and / or in the production of a human and / or animal food products. In additional embodiments, the present invention provides for uses of plants, plant parts, cells or seed of as described herein in a plant breeding method and / or in the production of hybrid seed or other propagatable material.

[0062] In embodiments of the invention, RNP complexes comprising the gRNAs described herein may be introduced into plant cells or protoplasts via techniques known to the person of skill in the art. For instance, RNP complexes may be formed in vitro and mixed directly with recipient protoplasts. Alternatively, plant cells or protoplasts may be transformed with one or more plasmid or viral vectors that express the Cas endonuclease and the gRNA in the cell.

[0063] The invention is further illustrated by the following non-limiting examples. EXAMPLES

[0064] Selection of commercial varieties of interest:

[0065] Four varieties of potato were selected for StPPO2 studies based on their production area and main uses.

[0066] Russet Burbank - The main variety produced in the United States, used in the food processing market and fresh market to a lesser extent.

[0067] Russet Norkotah -A variety grown on large number of acres in the United States, mainly used in the fresh potato market.

[0068] Atlantic - The most produced chip potato variety in the United States.

[0069] Variety “G” - A private variety that is cultivated in the United States and the Europe for the fresh market but which is known to have a high sensitivity to bruising.

[0070] Micro-plants for the selected varieties were obtained with full cooperation from the respective breeders, and by requesting micro-plants in tissue culture from Scottish Agricultural Science Agency. To the extent that it applies, genetic material was obtained in accordance with the requirements of the Nagoya Protocol with the country of origin identified as the United States of America and United Kingdom. :ic material:

[0071] Whole leaves were taken from each of the potato varieties and used for DNA extraction using Zymo Quick DNA Plant / Seed Extraction Kit by following the manufacturers protocol. To the extent that it applies, genetic material was obtained in accordance with the requirements of the Nagoya Protocol.

[0072] The published sequence of StPPO2 was used from Thygesen et al. (Plant Physiol. (1995) 109: 525-531) and available in the GenBank database (https: / / www.ncbi.nlm.nih.gov) under ID: U22921.1 (see Table 1) on chromosome 8. This sequence was used to design several primer pairs spanning the StPPO2 gene body. Then, DNA from each variety was used in a PCR to amplify fragments of StPPO2. The PCR product was purified and sent for NGS ampliconsequencing. Sequencing results were first used to determine how many alleles were being amplified from each primer pair by using the number and percentage of polymorphisms detected. The sequences of primer pairs that amplified for all four variety alleles were mapped against U22921.1 to detect conserved regions with low polymorphic changes in all varieties. Those regions were then compared inter-variety and one region of the StPPO2 was selected for gRNA design: 700-1200 base pairs from the start codon ATG of the U22921 .1 gene. Table 1 - U22921.1 Solanum tuberosum tuber polyphenol oxidase PPO (POT32 allele) mRNA, complete cds [SEQ ID NO: 1]

[0073] Target selection:

[0074] Targets for gene editing were selected using RGEN tool Cas Designer (http: / / www.rgenome.net), selecting MAD7 as the intended nuclease and Potato genome as the desired organism of study. The portion of 700-1200 bp from ID: U22921.1 sequence was used as the input to obtain sgRNA options. The sgRNA were analysed for the amount of possible off- targets and only sgRNAs that showed a single target (StPP02) with up to 2 miss-matches in the Potato genome assembly DM_1-3_516_R44_potato_genome_assembly.v6.1 were selected for further studies. A range of five gRNA candidates were selected covering the most conserved parts of the identified portion of the gene (see Figure 1). The alleles of G (private) potato variety were used as a starting point to determine if gRNAs would bind to all the four alleles and three gRNAs were selected as particularly suitable: sgRNA_6, sgRNA_14, and sgRNA_28. The target binding sequences of sgRNA_6, sgRNA_14, and sgRNA_28 are set out in Table 2 below.

[0075] Table 2 - Solanum tuberosum tuber polyphenol oxidase PPO (POT32 allele) targeting sgRNAs, [SEQ ID NOs: 2-4]

[0076] Protoplast isolation and transfection:

[0077] Protoplasts were isolated from young leaves of potato plantlets grown in tissue culture conditions. Leaves were sliced into small strips and incubated in a media to promote cell plasmolysis. Then, the plasmolysis media was replaced by an enzymatic solution to promote cell wall degradation. When sufficient digestion of the cell walls occurred, the solution containing free cells (i.e. protoplasts) was collected and filtered several times to obtain solely wealthy protoplasts. The protoplasts were counted and diluted at a specific density in a conductive media for transfection. In parallel, the ribonucleoprotein (RNP) was prepared by combining a pre-determined amount of ErCas12a and sgRNA in a buffered solution that was left to incubate until needed. The RNP was added to 1 volume of diluted protoplasts followed by 1 volume of PEG solution. The liquids were homogenised and left to incubate for several minutes followed by washing steps with a specific media. After that, protoplasts were resuspended in regeneration solution A at a specific density.

[0078] Protoplast regeneration:

[0079] Protoplasts in regeneration solution A were mixed with 1 volume of alginate solution and plated in a media containing calcium to promote the hardening of alginate. The alginate matrix containing the protoplasts was incubated for several days in regeneration solution A. At a suitable developmental stage, regeneration solution A was replaced by regeneration solution B. Protoplasts developed into individual calli that were released from the alginate matrix and spread over regeneration solution B. After several days of incubation large calli were moved to regeneration media C and then D to promote shooting. Shoots were collected into regeneration media E to form roots. After several days fully developed plantlets were obtained. qRNA efficiency in-planta

[0080] The three gRNAs were used in a ribonucleoprotein (RNP) complex in G (private) protoplast transfection mediated by PEG. Samples were collected after 7 days for DNA extraction, PCR amplification and purification, and sent for NGS amplicon-seq. gRNA efficiency was measured by the % of edited reads on the target site compared with reads containing the native sequence. sgRNA_14 and sgRNA_28 (SEQ ID Nos: 3 and 4) showed the highest gene editing efficiency (see Figure 2) and therefore were used for gene editing in the other tested varieties. qRNA efficiency across multiple varieties:

[0081] The location of sgRNA_14 and sgRNA_28 (SEQ ID Nos: 3 and 4) was compared across the potato plant varieties G, Atlantic (A), Russet Burbank (RB), and Russet Norkotah (RN). The results are shown in Figure 3. sgRNA_28 (SEQ ID NO: 4) was identified as being in a conserved region without detectable polymorphisms in any of the plant varieties. sgRNA_14 (SEQ ID NO: 3) location was polymorphic, with some varieties displaying a single nucleotide substitution (single nucleotide polymorphism - SNP) in 2 alleles from Atlantic and Russet Burbank, whereas Russet Norkotah had one SNP in one allele. Protoplasts were isolated and transfected using sgRNA_14 and sgRNA_28 to test the efficiency of the gRNAs across varieties and to test the effect of the SNP in these varieties compared with G. Samples were collected after seven days for DNA extraction, PCR amplification and purification, and sent for NGS amplicon-seq. It was found the surprisingly, the sgRNA_28 gene editing efficiency was comparable among all the four tested varieties (G, RB, A, RN) in the alleles containing a SNP (see Figure 3). Without wishing to be bound by theory, the higher level of gene editing seen in sgRNA_14 in variety G (private) compared to other varieties may be correlated with an increased number of SNPs present in the target sequences in Russet Burbank (2), Russet Norkotah (1), and Atlantic (2).

[0082] For an sgRNA_28 modified plant, reads were obtained by next generation sequencing (NGS) and were mapped against the wild type PPO2 allele to characterise the indels resulting from the gene editing. As shown in Figure 4, the reference wild type allele (SEQ ID NO: 5), with location of the spacer sequence highlighted, is aligned to three modified alleles (SEQ ID NOs: 6-8) that exhibit deletions of 4, 10, and 8 nucleotides respectively. Based on the NGS read frequency mapping, this individual plant has 2 alleles both lacking 4 nucleotides (SEQ ID NO: 6), and 2 alleles lacking 8 or 10 nucleotides each (SEQ ID Nos: 7 and 8).

[0083] Phenotypic confirmation of trait implementation:

[0084] Fully grown potato plants were regenerated from tissue culture and moved to soil in a glasshouse to produce tubers (see Figure 5). Tubers were collected and sliced to expose the tissue to the oxygen and monitored throughout 48 hours at periodic intervals. The colour of the tuber was assessed visually at set timepoints to determine if the gene editing of the StPPO2 was sufficient to significantly reduce or eliminate oxidative browning in comparison to the unmodified control variety. varieties:

[0085] To assess the impact of StPPO2 knock-out on tuber browning, the potato varieties “G”, Atlantic, and Russet Burbank (original varieties) were used to produce gene-edited lines with complete StPPO2 knock-out (improved varieties). Plants from both groups were grown in a glasshouse, and tubers were harvested for browning analysis.

[0086] Browning, triggered by mechanical stress, was induced by peeling and grating tubers to maximize cell damage. Images were captured over a 14-hour period to monitor the browning process. By the end of this period, original varieties displayed an almost black colour due to melanin production, while improved varieties showed negligible browning. These results confirm that mutating all four StPPO2 alleles in the tested varieties effectively eliminates browning in response to damage. The results of this screening are shown in Figures 6A-C.

[0087] Growth comparison of plants from original and improved lines in a glasshouse:

[0088] To evaluate whether gene editing of StPPO2 and regeneration of whole plants from protoplasts affected plant growth and yield, original Variety “G” lines and improved lines (with StPPO2 knock-out) were grown side-by-side in a glasshouse. Plants from both groups were cultivated in tissue culture media for 30 days before being transplanted into soil in a glasshouse environment.

[0089] For each line, 48 plants were grown, distributed across four trays, each containing 12 plants. After 45 days, above-ground fresh biomass and tuber weights were collected and compared between the original Variety “G” line and improved lines. Data was visualized in bar plots to contrast the traits between the lines (Figures 7A and 7B). The results indicated no significant differences between the original and improved lines, demonstrating that StPPO2 knock-out and regeneration from protoplasts do not negatively impact plant development or yield.

[0090] Enzymatic activity of StPPO2 in potato tubers from original and improved varieties:

[0091] In addition to visual browning assessment, StPPO2 enzymatic activity was evaluated. Tubers from both original and improved lines were randomly selected, and cylindrical samples were extracted using a cork borer. Thin slices (2 mm) were incubated in a buffer solution containing L-DOPA, a substrate for PPO2. Control slices were incubated in buffer without L-DOPA to account for background signal. After 12 minutes, the enzymatic activity of StPPO2 was accessed indirectly by comparing the buffer colour change due to conversion of L-DOPA to melanin. Pictures were of the buffer of each sample were used to quantify pixel intensity using Imaged software. Background signals were subtracted to determine relative intensity. Comparison of the colour change in original and improved lines reveals significantly lower enzymatic activity in improved lines compared to original. Results are shown in Figure 8. Specifically, Variety “G” improved lines showed 2-3 times lower activity (Figure 8A), while improved Atlantic (Figure 8B) and Russet Burbank (Figure 8C) lines exhibited reductions of 10- 15 times. These results indicate that by knocking-out StPPO2, the capacity to produce melanin from L-DOPA is removed, which justifies the non-browning phenotype of tubers.

[0092] Enzymatic activity of StPPO2 in Variety “G” potato tubers from original and improved lines containing in-frame mutations:

[0093] The enzymatic activity of StPPO2 was also analysed in Variety “G” improved lines containing one allele with an in-frame mutation and three alleles with out-of-frame mutations. These lines displayed activity levels comparable to those with four out-of-frame mutations, indicating that sg28 (i.e. sgRNA_28) induces deletions of key amino acids critical for StPPO2-mediated oxidation. Results are shown in Figure 9.

[0094] PPG activity may also by determined enzymatically in homogenised tissue extracts by measuring the initial rate of oxygen uptake at 25°C in 50 mM sodium phosphate, pH 6.0. The reaction is initiated by the addition of the substrate 4-methyl catechol to a final concentration of 2 mM. One unit of activity is defined as that which catalyzes the consumption of 1 pmol of oxygen per minute under the assay conditions.

[0095] Gene editing efficiency of sq28 in different potato varieties:

[0096] To achieve gene editing of StPPO2, a CRISPR-Cas system was employed. Cas12 binding sites were identified in the target gene, prioritizing regions in the middle of the gene where nucleotide insertions or deletions are most effective at achieving gene knock-out. Multiple guide RNAs were evaluated in vitro and in vivo. Among them, sg28 (i.e. sgRNA_28) exhibited the highest activity and was further tested across different potato varieties.

[0097] To assess the efficiency of sg28 in editing StPPO2 in protoplasts from various potato varieties, the sg28 guide RNA was combined with E.r.Cas12 to form a ribonucleoprotein (RNP) complex. This complex was transfected into protoplasts from five potato varieties: Variety “G”, Atlantic, Russet Burbank, Russet Norkotah, and Ranger Russet. Post-transfection, protoplasts were embedded in a matrix solution and incubated for seven days. DNA was extracted, and the target region was amplified via PCR for amplicon sequencing. Editing efficiency was calculated using CRISPRESSO based on next-generation sequencing (NGS) data. Multiple biological replicates were analysed for each variety. The results showed that sg28 achieved gene editing efficiency exceeding 30% across all tested varieties. The results are shown in Figure 10. Although particular embodiments of the invention have been disclosed herein in detail, this has been done by way of example and for the purposes of illustration only. The aforementioned embodiments are not intended to be limiting with respect to the scope of the appended claims, which follow. The choice of nucleic acid starting material, the clone of interest, or type of library used is believed to be a routine matter for the person of skill in the art with knowledge of the presently described embodiments. It is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims.

Claims

WHAT IS CLAIMED IS:

1. A Solanum tuberosum plant cell comprising a mutation in at least one allele of a polyphenol oxidase 2 (StPP02) gene within a region 700-1200 base pairs from the ATG start codon of the StPPO2 gene, wherein the mutation generates a phenotype of reduced or eliminated polyphenol oxidase activity when compared to a Solanum tuberosum plant cell without the mutation.

2. The plant cell of claim 1 , wherein the mutation in at least one allele of the StPPO2 gene is within a region 700-800 base pairs from the ATG start codon of the StPPO2 gene.

3. The plant cell of claim 1 , wherein the mutation in at least one allele of the StPPO2 gene is within a region 1100-1200 base pairs from the ATG start codon of the StPPO2 gene.

4. The plant cell of claim 1 , wherein the mutation is comprised within multiple alleles of the StPPO2 gene.

5. The plant cell of claim 4, wherein the mutation is comprised within a region 700-800 base pairs and a region 1100-1200 base pairs from the ATG start codon of the StPPO2 gene.

6. The plant cell of any one of claims 1 to 5, wherein the StPPO2 gene comprises one or more alleles comprising a sequence according to SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

7. The plant cell of any one of claims 1 to 6, wherein the mutation is a frameshift mutation suitably the result of an insertion during non-homologous end joining (NHEJ) repair or homology directed repair (HDR) following a staggered double strand break in the DNA.

8. A potato tuber comprising a plant cell of any one of claims 1 to 7.

9. The potato tuber of claim 8, wherein the tuber is resistant to PPG mediated browning reactions.

10. The potato tuber of claim 9, wherein the tuber exhibits reduced PPO mediated browning reactions compared to a non-genetically modified potato tuber of the same variety or species.11 . The potato tuber of any one of claims 8 to 10, wherein tuber is derived from a Solanum tuberosum variety is selected from: Abbot, Accent, Adirondack Blue, Adirondack Red, Agata, Agria, Almond, Alturas, Amandine, Americar, Amin, Annabelle, Anya, Arran Victory, Atlantic,Arizona, Austrian Crescent, Bamberg, Belana, Belle de Fontenay, BF-15, Bildtstar, Bintje, Blackberry, Blue Congo, Blue Danube, Bonnotte, Canela Russet, Cara, Caribou Russett, Cabritas, Camota, Cardinal, Centennial Russet, Challanger, Charlotte, Chelina, Chieftain, Chiloe, Cielo, Ciklamen, Clavela Blanca, Clearwater Russet, Colleen, Congo, Cosmos, Dakota Russett, Desiree, Draga, Duke of York, Edzell Blue, Elba, Faraja, Feldeslohn, Fianna, Fingerling, Flava, Fontana, Frisia, Gala, Georgina, German Butterball, Gold Rush, Golden Wonder, Golof, Hannah Sweet, Hunter, Innovator, Japanese Sweet, Jersey Royal, Kerr”s Pink, Kennebec, Kenya Baraka, Kestrel, King Edward, Kinongo, Kipfler, Kuras, Lady Balfour, Lady Crystal, Lady Felicia, Lady Rosetta, Lamoka, Laura, Linda, Magic Molly, Marfona, Marilynn, Maris Bard, Maris Piper, Maris Peer, Marquis, Mellody, Meru Mix, Moonlight, Mozart, Mukorino, Nadine, Natascha, Nectar, Ngure, Nicola, Norland, Novello, Nyayo Mukori, Orla, Pachacona, Patrones, Pimpernel, Pink Eye, Pink Fir Apple, Pinto Gold, Pirol, Primura, Ratte, Picasso, Purple Majesty, Purple Viking, Purplu, Ranger Russet, Red Gold, Red Norland, Red Pontiac, Red Rascal, Red Thumb, Reveille Russet, Robijn, Robinta, Rockstar, Romano, Rooster, Roslin Chania, Roslin Tana, Roslin, Royal, Rudolph, Rua, Rugano, Russet Burbank (selections), Russet Norkotah (selections), Sante, Sarpo Mira, Sasamua Satina, Selma, Setanta, Shepody, Sieglinde, Sirco, Soraya, Spunta, Snowden, Stobrawa, Strawberry Paw, Umatilla Russet, Urgentia, Valor, Vivaldi, Vitelotte, Waneta, Wilja, Yellow Finn, or Yukon Gold.

12. A Solanum tuberosum plant or plant part thereof comprising a plant cell of any one of claims 1 to 7, wherein the plant exhibits a phenotype of reduced PPG mediated browning reactions compared to a non-genetically modified Solanum tuberosum plant.

13. The plant of claim 12, wherein the plant part is selected from the group consisting of a leaf, pollen, an ovule, a fruit, rootstock, a scion, a tuber, a flower, and a cell.

14. A tissue culture of regenerable cells of the plant or part thereof of either of claims 12 or 13.

15. A Cas12a ribonucleoprotein (RNP) complex comprising a Cas12a endonuclease and a guide RNA (gRNA) that hybridises with a target sequence comprised within a polyphenol oxidase 2 (StPPO2) gene of a Solanum tuberosum variety.

16. The RNP complex of claim 15, wherein the gRNA hybridises with a target sequence comprised within a region of a StPPO2 gene defined as 700-1200 base pairs downstream from the ATG start codon of the StPPO2 gene.

17. The RNP complex of claim 16, wherein the gRNA comprises a sequence selected from one of SEQ ID Nos: 2-4.

18. The RNP complex of claim 17, wherein the gRNA comprises a sequence of SEQ ID NO: 4.

19. The RNP complex of any one of claims 15 to 18, wherein the Cas12a endonuclease is a MAD7 endonuclease.

20. The RNP complex of any one of claims 15 to 19, wherein the gRNA hybridises with a target sequence comprised within a StPPO2 gene of at least two Solanum tuberosum varieties.

21. The RNP complex of any one of claims 15 to 20, wherein the Solanum tuberosum variety is selected from: Abbot, Accent, Adirondack Blue, Adirondack Red, Agata, Agria, Almond, Alturas, Amandine, Americar, Amin, Annabelle, Anya, Arran Victory, Atlantic, Arizona, Austrian Crescent, Bamberg, Belana, Belle de Fontenay, BF-15, Bildtstar, Bintje, Blackberry, Blue Congo, Blue Danube, Bonnotte, Canela Russet, Cara, Caribou Russett, Cabritas, Camota, Cardinal, Centennial Russet, Challanger, Charlotte, Chelina, Chieftain, Chiloe, Cielo, Ciklamen, Clavela Blanca, Clearwater Russet, Colleen, Congo, Cosmos, Dakota Russett, Desiree, Draga, Duke of York, Edzell Blue, Elba, Faraja, Feldeslohn, Fianna, Fingerling, Flava, Fontana, Frisia, Gala, Georgina, German Butterball, Gold Rush, Golden Wonder, Golof, Hannah Sweet, Hunter, Innovator, Japanese Sweet, Jersey Royal, Kerr”s Pink, Kennebec, Kenya Baraka, Kestrel, King Edward, Kinongo, Kipfler, Kuras, Lady Balfour, Lady Crystal, Lady Felicia, Lady Rosetta, Lamoka, Laura, Linda, Magic Molly, Marfona, Marilynn, Maris Bard, Maris Piper, Maris Peer, Marquis, Mellody, Meru Mix, Moonlight, Mozart, Mukorino, Nadine, Natascha, Nectar, Ngure, Nicola, Norland, Novello, Nyayo Mukori, Orla, Pachacona, Patrones, Pimpernel, Pink Eye, Pink Fir Apple, Pinto Gold, Pirol, Primura, Ratte, Picasso, Purple Majesty, Purple Viking, Purplu, Ranger Russet, Red Gold, Red Norland, Red Pontiac, Red Rascal, Red Thumb, Reveille Russet, Robijn, Robinta, Rockstar, Romano, Rooster, Roslin Chania, Roslin Tana, Roslin, Royal, Rudolph, Rua, Rugano, Russet Burbank (selections), Russet Norkotah (selections), Sante, Sarpo Mira, Sasamua Satina, Selma, Setanta, Shepody, Sieglinde, Sirco, Soraya, Spunta, Snowden, Stobrawa, Strawberry Paw, Umatilla Russet, Urgentia, Valor, Vivaldi, Vitelotte, Waneta, Wilja, Yellow Finn, or Yukon Gold.

22. The plant cell of claim 7, wherein the staggered double strand break in the DNA is due to gene editing utilising an RNP complex of any one of claims 15 to 21 .

23. An isolated guide RNA comprising a nucleotide sequence selected from any one of SEQ ID Nos: 2-4.

24. The isolated guide RNA sequence of claim 23, wherein the guide RNA is adapted for use with a Cas12a endonuclease.

25. The isolated guide RNA sequence of claim 24, wherein the Cas12a endonuclease is MAD7.

26. A method for reducing or eliminating PPO activity in a plant cell from a Solanum tuberosum species, the method comprising mutating one or more alleles of an StPPO2 gene within the genome of the plant cell, wherein the mutation is a frameshift mutation resulting from an insertion or deletion located within a region 700-1200 base pairs from an ATG start codon of the StPPO2 gene, and wherein the mutation is the result of a CRISPR / Cas gene editing event.

27. The method of claim 26, wherein the mutation is a frameshift mutation resulting from an insertion or deletion located within a region 700-800 base pairs from an ATG start codon of the StPPO2 gene.

28. The method of claim 26, wherein the mutation is a frameshift mutation resulting from an insertion or deletion located within a region 1100-1200 base pairs from an ATG start codon of the StPPO2 gene.

29. The method of claim 26, wherein the mutation is a frameshift mutation resulting from an insertion or deletion located within a region 700-800 base pairs and an insertion or deletion located within a region 1100-1200 base pairs from an ATG start codon of the StPPO2 gene.

30. The method of any one of claims 26 to 29, wherein the CRISPR / Cas gene editing event is the result of a CRISPR / Cas12a directed gene editing event utilising an RNP as described in any one of claims 15 to 21.31 . The method of any one of claims 26 to 30, wherein the method comprises regenerating a whole plant from the plant cell, wherein the whole plant exhibits a phenotype of reduced or eliminated PPO activity when compared to a Solanum tuberosum plant not having the mutation.

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