Potatoes with reduced polyphenol oxidase activity

Abstract

This invention relates to reducing potato (…) using endonuclease-directed gene editing technology. Solanum tuberosum Improvements to tuber browning in potato varieties. Specifically, the present invention provides potato plant cells containing a mutant, which, compared to potato plant cells without the mutant, produces a phenotype of reduced or eliminated polyphenol oxidase activity. The present invention also provides a ribonucleoprotein complex and a method of using the same, the ribonucleoprotein complex comprising a nuclease and polyphenol oxidase 2 contained in a potato variety. StPPO2 Guide RNA for hybridization to target sequences within genes.

Description

Technical Field

[0001] This invention relates to the use of endonuclease-mediated gene editing technology to improve potato ( Solanum tuberosum Varieties to reduce tuber browning. Background Technology

[0002] Originating in western South America, the potato grows naturally in the wild, exhibiting the widest diversity in tuber shape, size, color, and taste. After being brought to Europe by Spanish conquistadors in the latter half of the 16th century, the plant became extremely important and subsequently spread throughout the world along global trade routes. Ultimately, the potato became the most widely cultivated tuber crop and the fourth most important food crop globally, after wheat, rice, and corn. Its ease of cultivation, lower land requirements compared to other major food crops, and high nutritional value make it a staple crop in many countries. Advances in agronomy and biotechnology have bred thousands of diverse cultivars and varieties, enabling increased yields and better adaptation to climate stresses.

[0003] Potatoes belong to the genus *Solanum* in the family Solanaceae and have 12 basic chromosomes. Potatoes are not only a widely used vegetable but also used to make various processed foods. They are also used industrially to produce starch, alcoholic beverages, and even biofuels. Developing varieties with important agronomical traits and good storage properties is one of the main challenges facing potato breeders.

[0004] Polyphenol oxidase (PPO), typically encoded by a multi-gene family, causes oxidative browning, a serious problem in many foods, particularly potato tubers. PPO is virtually ubiquitous in angiosperms and belongs to a class of copper-binding enzymes that catalyze the oxidation of phenolic compounds into quinones. The subsequent non-enzymatic polymerization of quinones leads to the formation of brown pigments, sometimes called "black spots," which contribute to post-harvest spoilage (such as bruising) and deterioration in the quality of stored potatoes. Losses due to PPO-catalyzed oxidation and browning account for 50% of production losses in the fruit and vegetable industry. It is estimated that in 1994 alone, the total economic loss from potato bruising in the United States reached $298.9 million (Brook (1996), "Potato Bruising – How and Why Emphasising Black Spot Bruise," Running Water Publishing, ISBN: 0-9650498-0-9). By 2025, the economic impact of potato bruising in the United States could exceed $600 million. Besides the undesirable browning activity, members of the PPO gene family appear to play important roles in signal transduction, stress response, and defense responses throughout plant growth and development. Therefore, simply knocking out all PPO genes is not a feasible method for producing improved potato varieties.

[0005] US-8889964-B1 describes a novel potato variety called “W8”, transformed using a natural nucleic acid sequence from the potato plant genome. The DNA inserted into the genome of potato variety W8 is a non-coding polynucleotide that silences genes associated with black spot bruising, asparagine accumulation, and senescent sweetening phenotypes. The patent describes a plant vector called pSIM1278, comprising a first and a second silence box. The first silence box contains two copies of a DNA fragment containing, in the antisense direction, a fragment of the asparagine synthase-1 gene (fAsn1) and a 3'-untranslated sequence of the polyphenol oxidase-5 gene; the second silence box contains two copies of a DNA fragment containing, in the antisense direction, a fragment of the potato phosphorylase-1 (pPhL) gene and a fragment of the potato R1 gene. Thus, tubers from potato plant varieties expressing at least one silence box exhibit two or more desired traits not present in tubers from untransformed plants of the same variety.

[0006] WO-2018 / 035456-A1 describes a potato plant, plant part, or plant cell containing a deletion mutation in at least one endogenous PPO allele, resulting in reduced PPO expression compared to control potato plants. Mutation was performed using a transcription activator-like effector endonuclease (TALE-nuclease) targeting the POT32 or POT33 allele.

[0007] González et al. (Plant Cell Tiss Organ Cult 145, 291–305 (2021)) described Agrobacterium-mediated transformation and protoplast transfection delivery targeting the potato variety Desiree. StPPO2 The CRISPR / Cas9 component of the gene. Two sgRNAs were designed to simultaneously target Cas9. StPPO2 The authors report that the dual-sgRNA strategy resulted in poor performance due to the low incidence of targeted deletions. Furthermore, reliance on a dual-sgRNA strategy increases the risk of unintended modifications, such as off-target mutations and / or large deletions at target sites. Additionally, González et al. point out that sgRNAs are produced by DNA molecules, rather than through chemical synthesis, which further increases the likelihood of them generating unintended insertions in the genome.

[0008] Therefore, a greater variety of options is still needed to obtain the non-browning phenotype in potatoes. A new set of allelic variants is still needed to confer the non-browning phenotype in a wide range of potato varieties. This is particularly important because different specialty potato varieties are used for different purposes, such as processed foods, potato chips, French fries, or fresh potatoes.

[0009] Based on the teachings provided herein, these and other uses, features, and advantages of the invention will be apparent to those skilled in the art. Summary of the Invention

[0010] In a first aspect of the invention, a potato plant cell is provided, comprising: StPPO2 A mutation in at least one allele of a gene, said mutation being at a distance from... StPPO2 Within the 700-1200 base pair region of the ATG start codon, the mutation produces a phenotype of reduced or eliminated polyphenol oxidase activity compared to potato plant cells without the mutation.

[0011] A second aspect of the invention provides a Cas12a ribonucleoprotein (RNP) complex comprising a Cas12a endonuclease and a polyphenol oxidase 2 contained in a potato variety. StPPO2 Guide RNA (gRNA) that hybridizes to a target sequence within a gene. In embodiments of the invention, the gRNA hybridizes with a sequence contained in one or more potato varieties (typically at least two, three, or more varieties). StPPO2 Hybridization of target sequences within genes.

[0012] A third aspect of the invention provides potato tubers comprising the plant cells described herein. Suitably, the potato tubers are resistant to PPO-mediated browning.

[0013] A fourth aspect of the invention provides a potato plant or a plant portion thereof comprising the plant cells described herein, wherein the plant exhibits a phenotype of reduced browning response mediated by PPO compared to non-transgenic potato plants.

[0014] A fifth aspect of the invention provides tissue cultures of regenerative cells of plants or plant parts thereof as described herein.

[0015] A sixth aspect of the present invention provides an isolated guide RNA comprising a nucleotide sequence selected from any one of SEQ ID NO: 2-4.

[0016] A seventh aspect provides a method for reducing or eliminating PPO activity from plant cells of a potato species, the method comprising targeting the genome of the plant cells... StPPO2One or more alleles of a gene are mutated, wherein the mutation is caused by a gene located at a distance from the gene. StPPO2 A frameshift mutation is caused by an insertion or deletion within the 700-1200 base pair region of the ATG start codon of a gene, and said mutation is the result of a CRISPR / Cas gene editing event.

[0017] Within the scope of this disclosure, it is expressly intended that the various aspects, embodiments, examples, and alternatives set forth in the foregoing paragraphs, claims, and / or the following description and drawings, in particular their individual features, may be employed independently or in any combination. That is, all embodiments and / or features of any embodiment may be combined in any manner and / or combination unless such features are incompatible. Attached Figure Description

[0018] Now, one or more embodiments of the invention will be described by way of example only, with reference to the accompanying drawings, wherein:

[0019] Figure 1 The aligned allele sequences from four potato varieties are shown to identify conserved regions among the varieties. This allows for the selection of appropriate exemplary sgRNAs based on perfect or near-perfect matches with the identification sequences in each variety. The figure shows the location and 5'-3' orientation of a series of tested sgRNAs. The vertical rectangular bars in sgRNA_34 and sgRNA_14 represent single nucleotide polymorphisms (SNPs) compared to the potato reference genome.

[0020] Figure 2 It is a display Figure 1 Five sgRNAs identified in the study were transfected into protoplasts in a proprietary potato variety named "G". StPPO2 A graph of gene editing efficiency (%GE).

[0021] Figure 3 This demonstrates protoplast transfection using guide RNAs sgRNA_14 and sgRNA_28 via PEG transfection and Cas12a (ErCas12a) ribonucleoprotein in four potato varieties. StPPO2 Efficiency of gene editing (%GE).

[0022] Figure 4 The diagram shows the sgRNA_28 guide RNA and its representation of the wild type. StPPO2 Sequence alignment of the reference strand of the allele location (SEQ ID NO: 5), and edited modified alleles (SEQ ID NO: 6-8) obtained from single plants with deletions of 4, 10 and 8 nucleotides, respectively.

[0023] Figure 5 The image shows a photograph of a gene-edited potato plant regenerated through tissue culture.

[0024] Figure 6 shows the results of screening potato tuber browning phenotypes from primitive and improved varieties. Figure 6A These are photos of tubers from the original and improved varieties of Atlantic and Rust Burbank after they have been crushed and incubated for 14 hours. Figure 6B The photos show four incubation time points after the original and improved variety “G” tubers were ground. Figure 6C These are photos of the original and improved variety "G" tubers ground and incubated for 14 hours.

[0025] Figure 7 shows the average leaf weight of the original (unedited) and improved (edited) potato lines. Figure 7A ) and tuber weight ( Figure 7B (The image is shown.)

[0026] Figure 8 shows the enzyme assay results for non-browning in the original and improved potato lines. Figure 8A This is a graph showing the PPO enzyme activity results in the original and improved "G" line varieties. Figure 8B This is a graph showing the PPO enzyme activity results in the original and modified Atlantic lines. Figure 8C This is a graph showing the PPO enzyme activity results in the original and modified Russet Burbank lines.

[0027] Figure 9 The images show potato tubers from the original line and the improved line "G" containing missing values ​​of varying lengths within the frames. StPPO2 A graph showing the results of enzyme activity assay.

[0028] Figure 10 The graph shows the gene editing efficiency of sgRNA_28 (i.e., sg28) in different potato varieties. Detailed Implementation

[0029] All references cited herein are incorporated herein by reference. 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 pertains. Before describing the invention, some definitions are provided to aid in understanding it.

[0030] Unless otherwise stated, the present invention is carried out using conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA technology and chemical methods, which are all within the capabilities of those skilled in the art. These techniques are also explained in the literature, for example, MR Green, J. Sambrook, 2012, Molecular Cloning: A Laboratory Manual, Fourth Edition, Books 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel, FM 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; JM Polak and James O'D. McGee, 1990, In Situ Hybridisation: Principles and Practice, Oxford University Press; MJ Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, IRL Press; and DMJ Lilley and JEDahlberg, 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, pp. 2-662 (2011); *Synthetic Biology, Part B, Computer Aided Design and DNA Assembly, Methods in Enzymology*, Edited by Christopher Voigt, Volume 498, pp. 2-500 (2011); *RNA Interference, Methods in Enzymology*, David R. Engelke and John J. Rossi, Volume 392, pp. 1-454 (2005). All references cited herein are incorporated by way of 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 pertains.

[0031] As used herein, the term "comprising" means that any of the listed elements must be included, and other elements may optionally be included. "Substantially consisting of" means that any of the listed elements must be included, excluding elements that would substantially affect the nature and novelty of the listed elements, and other elements may optionally be included. "Constitutes of" means excluding all elements other than those listed. Embodiments defined by each of these terms are within the scope of this invention.

[0032] A polynucleotide is a single-stranded or double-stranded covalently linked sequence of nucleotides, in which the 3' and 5' ends of each nucleotide are linked by a phosphodiester bond. Polynucleotides can consist of deoxyribonucleotide bases or ribonucleotide bases. Polynucleotides include DNA and RNA and can be synthesized in vitro or isolated from natural sources. The size of a polynucleotide is usually expressed as the number of base pairs (bp) in a double-stranded polynucleotide, or as the number of nucleotides (nt) in the case of a single-stranded polynucleotide. One thousand bp or nt equals one thousand bases (kb). Polynucleotides shorter than approximately 40 nucleotides are often called oligonucleotides.

[0033] When applied to polynucleotide sequences, the term "isolated" indicates that the sequence has been removed from the natural organism from which it originates, and therefore does not contain foreign or unwanted coding or regulatory sequences. Isolated oligonucleotides can be wholly or partially synthetic, i.e., chemically synthesized, rather than derived from a natural source.

[0034] When applied to peptide or ribonucleoprotein complexes, the adjective "isolated" refers to a substantially purified composition, or, in the case of a ribonucleoprotein complex, at least one component is substantially purified. Further, with respect to isolated ribonucleoprotein complexes, it is preferable that substantially all components are purified.

[0035] As used herein, the terms 3' ('3 prime') and 5' ('5 prime') take their usual meaning in the art, namely, distinguishing the ends or orientation within a polynucleotide sequence. Polynucleotides have 5' and 3' ends, and polynucleotide sequences are typically written in a 5' to 3' orientation. The 5' end is properly considered to be upstream of the 3' end of the polynucleotide sequence. Thus, a sequence referred to as upstream of a given reference point in a gene (such as the ATG transcription start codon in an open reading frame (ORF)) is a sequence located at the 5' reference point. Similarly, a sequence referred to as downstream is a sequence located at the 3' reference point, such as downstream of the ATG start codon.

[0036] As used herein, the terms “plant” and “plant part” refer to cells, tissues, organs, seeds, and cut portions (e.g., roots, leaves, and flowers) that retain the distinguishing characteristics of a parent plant. “Tuber” refers to the thickened underground portion of a stem or rhizome that serves as a food reserve and a bud for producing new plants. “Seed” refers to any plant structure that forms after the normal point of maturity at the time of flowering, through the continuous differentiation of the ovule, regardless of whether fertilization is present or absent, and regardless of whether the seed structure is fertile or sterile. Other reproductive parts of a plant may include tissue cultures of regenerable cells, callus, cuttings, or root segments. Suitably, “plant cell” may be selected from gametophyte, reproductive cells, vegetative cells, and / or meristematic cells. In the implementation scheme, suitable plant tissues are selected from: leaves, stems, roots, tubers, seeds, branches, short hairs, root nodules, leaf axils, flowers, pollen, stamens, pistils, petals, peduncles, stigmas, styles, bracts, fruits, trunks, carpels, sepals, anthers, ovules, pedicels, needles, cones, rhizomes, stolons, young branches, pericarps, endosperm, placentations, berries, stamens, or leaf sheaths. In a particular implementation scheme, plant cellular material may include root tissue, mesophyll, and / or cultured callus.

[0037] In some embodiments, the plant cells are in the form of protoplasts. As used herein, the term "plant protoplast" (also simply "protoplast" in this disclosure) refers to a plant cell whose cell wall has been completely or partially removed. Cell wall removal can be achieved by mechanical, chemical, or enzymatic methods. In embodiments, cell wall digestive enzymes are used to obtain protoplasts from suitable plant material. For example, enzymes such as cellulase, macrozyme, pectinase, hemicellulase, pectolyase, driselase, xylanase, and combinations thereof are suitable for use in the context of this invention. In embodiments, cellulase can be used at a concentration of 1 wt% to 1.5 wt%. In embodiments, macrozyme can be used at a concentration of 0.2 wt% to 0.4 wt%. In embodiments, hemicellulase can be used at a concentration of 2 wt% to 5 wt%. In embodiments, pectolyase can be used at a concentration of 0.01 wt% to 0.5 wt%. In the implementation, the collapse enzyme can be used at a concentration of 0.5 w% - 2 w%. Protocols for obtaining protoplasts from plant tissues are known in the art, for example in Yoo, Cho, and Sheen (2007) Nature Protocols, Vol. 2, pp. 1565-1572.

[0038] The term "allele" refers to one or more alternative forms of a gene at a specific locus. In the diploid (or double diploid) cells of an organism, the allele of a given gene is located at a specific location or locus on a chromosome, with one allele on each of each pair of homologous chromosomes. Similarly, in the tetraploid cells of an organism, one allele is located on each of the four homologous chromosomes in a set of organisms. "Heterozygous" alleles are different alleles located at a specific locus, each on its corresponding homologous chromosome. "Homozygous" alleles are identical alleles located at a specific locus, each on its corresponding homologous chromosome in the cell.

[0039] The term "wild type" as used in this article refers to the most common plant or gene form in nature.

[0040] The term "allelic variant" is used herein to refer to any two or more alternative forms of a gene that occupies the same chromosomal locus and controls the same genetic trait. Allelic variants arise naturally through mutation and can lead to phenotypic polymorphism within a population. Gene mutations typically result in changes to the nucleic acid sequence and, in some cases, changes to the polypeptide sequence. As used herein, the term "allelic variant" also refers to a protein or polypeptide encoded by an allelic variant of a gene.

[0041] The "wild-type PPO2 allele" is a naturally occurring PPO2 allele that encodes a functional PPO2 protein (such as in naturally occurring potatoes). S. tuberosum The gene identified in the plant (genus code PGSC0003DMG400018916) is a "non-functional mutant PPO2 allele," which is a PPO2 allele that does not encode a functional PPO2 protein. Such a "non-functional mutant PPO2 allele" may include one or more mutations in its nucleic acid sequence, said mutations resulting in a reduced or even undetectable amount of functional PPO2 protein in vivo or in plant cells, or in extracts taken from mutant plant cells and tested in vitro.

[0042] When used in relation to genes, the terms “elimination,” “inhibition,” or “reduction” refer to a decrease, reduction, or elimination of the expression level of a gene-encoded mRNA and / or protein product, and / or a decrease, reduction, or elimination of the activity of a gene-encoded protein in a plant, plant cell, or plant tissue, compared to the expression level of such target mRNA and / or protein in a wild-type or control plant, cell, or tissue at the same stage of plant development, and / or compared to the activity of such encoded proteins. The reduction, decrease, or elimination can be functionally determined, for example, by testing the activity of the gene product, or by various quantitative measurements, such as measurements assessing a reduction in protein product production (e.g., Western blotting, mass spectrometry, ELISA).

[0043] The genetically engineered or gene-edited potato cells or protoplasts described herein can be prepared using conventional gene-editing methods or methods more specifically described herein to edit one or more target PPO genes. Targeted editing can be achieved through non-nuclease-dependent or nuclease-dependent methods. In non-nuclease-dependent targeted editing methods, homologous recombination is guided by a homologous sequence flanking the exogenous polynucleotide to be introduced into the endogenous sequence, and proceeds through the enzymatic mechanisms of the host cell. The exogenous polynucleotide can be in the endogenous sequence, such as in... StPPO2 Nucleotide deletions, insertions, or substitutions are introduced into the recognition region of a gene. StPPO2 Mutations in genes can be used to completely knock out gene function and / or can produce allelic variants with reduced or impaired PPO activity.

[0044] Alternatively, nuclease-dependent methods can achieve targeted editing at a higher frequency by specifically introducing double-strand breaks (DSBs) using rare cleaving nucleases (e.g., endonucleases). This type of nuclease-dependent targeted editing also utilizes host DNA repair mechanisms, such as non-homologous end joining (NHEJ), which occurs in response to these DSBs. DNA repair via NHEJ can result in the random insertion or deletion (so-called "indels") of small amounts of endogenous nucleotides at the cleavage site. Repair can also occur via homology-directed repair (HDR), in contrast to NHEJ-mediated repair. When a donor template containing exogenous genetic material flanked by a pair of homologous arms is present, the exogenous genetic material can be introduced into the genome via HDR, leading to targeted integration of the exogenous genetic material.

[0045] In some implementations, gene disruption can occur by using two guide RNAs to delete genomic sequences. Nekrasov et al. described a method for creating genomic deletions in cells using CRISPR-Cas gene editing technology, such as gene knockout in plant cells, in Sci Rep 7, 482 (2017).

[0046] Available endonucleases capable of introducing specific and targeted DSBs include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided CRISPR-Cas nucleases (CRISPR-Cas; clustered regular-spaced short palindromic repeats). Furthermore, the DICE (dual integrase box exchange) system utilizing phiC31 and Bxb1 integrases can also be used for targeted integration.

[0047] The CRISPR-Cas system is a widely distributed prokaryotic adaptive immune system and is considered an integral part of next-generation genome editing tools. Cas9 recognizes 3' G-rich PAMs; it has become the most widely used CRISPR-Cas system and has been used for genome editing in a variety of contexts, including plants. Unlike Cas9, Cas12a recognizes 5' T-rich PAMs and self-processes its CRISPR RNA (crRNA). According to certain embodiments of the invention, an RNA-guided CRISPR-Cas12a nuclease (CRISPR / Cas12a) is used, preferably ErCas12a (MAD7) (US Patent No. US9982279 B1) (https: / / www.inscripta.com / madzymes / faq / ). MAD7 is an engineered nuclease of the type 2 VA CRISPR-Cas (Cas12a / Cpf1) family, with low homology to typical Cas12a nucleases, and is particularly effective in generating indel mutations in plant cells. Other non-restricted examples of RNA-guided endonuclease systems include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4, as well as their homologs or modified versions thereof.

[0048] CRISPR-Cas gene editing technology involves the use of genome-targeting nucleic acids that can direct an endonuclease to a specific target sequence within a target gene for gene editing at that specific target sequence. Genome-targeting nucleic acids can be RNA. Hereinafter, genome-targeting RNAs are referred to as “guide RNAs” or “gRNAs.” Guide RNAs typically contain at least one spacer (or protospacer) sequence and a CRISPR repeat sequence (crRNA) that hybridizes to a target nucleic acid sequence within the target gene to be edited. In type V gRNAs (e.g., ErCas12a / MAD7) utilized by the Cas12a nuclease, the crRNA forms a double-stranded structure that binds to the endonuclease, causing the guide RNA and endonuclease to form a complex. In some embodiments, the genome-targeting nucleic acid provides target specificity to the complex through its binding to the endonuclease. Thus, the genome-targeting nucleic acid directs the endonuclease activity to a specific target site within the host cell genome. Therefore, those skilled in the art will understand that each gRNA is designed to include a spacer sequence complementary to its genomic target sequence. The term guide RNA (gRNA) is synonymous with the single-stranded guide RNA “sgRNA.”

[0049] In embodiments of the invention, the spacer sequence contained in the gRNA is an oligonucleotide sequence, typically about 20 to 25 nucleotides in length, which defines the target sequence of the target gene (e.g., a DNA target sequence, such as a genomic target sequence). In embodiments of the invention, the target is... StPPO2 A region within a gene. In some embodiments, the spacer sequence is 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, the spacer sequence contains at least 22 nucleotides.

[0050] In an embodiment of the present invention, the selected "target sequence" is located in the target gene, and is preferably... StPPO2 A gene that is adjacent to a PAM sequence and is a sequence to be modified by an RNA-guided endonuclease (e.g., Cas12a or MAD7). In a specific embodiment of the invention, a gene is identified as having a suitable number of potential target sequences. StPPO2 Gene region distance StPPO2 The start codon (ATG) of a gene is 700-1200 base pairs long. In a specific embodiment of the invention, the target sequence is located at a distance from... StPPO2 The gene start codon (ATG) is 700-800 base pairs long. StPPO2 Gene region. In a specific embodiment of the present invention, the target sequence is located at a distance from... StPPO2 The gene start codon (ATG) is 1100-1200 base pairs long. StPPO2 Gene regions. In a specific embodiment of the present invention, the target sequence is derived from... StPPO2 Gene start codon (ATG) StPPO2 The gene consists of two target sequences in the 700-800 and 1100-1200 base pair regions. These "target sequences" are located on the so-called PAM-chain within the "target nucleic acid," which is a double-stranded DNA molecule containing both the PAM-chain and a complementary non-PAM chain. Those skilled in the art will recognize that the gRNA spacer sequence hybridizes with the complementary sequence located in the non-PAM chain of the target nucleic acid. The gRNA spacer sequence interacts with the target nucleic acid in a sequence-specific manner via Watson-Crick base pairing. Therefore, the nucleotide sequence of the spacer sequence varies depending on the target sequence of the target nucleic acid.

[0051] The gRNA disclosed herein can target any target sequence through the spacer sequence contained therein. In some embodiments, the 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 is 100% complementary to the target sequence in the target gene. In other embodiments, the spacer sequence of the guide RNA may contain up to five mismatches with the target sequence in the target gene, for example, up to four, up to three, up to two, or up to one mismatch. Typically, the mismatches are not consecutive and may be distributed across the spacer sequence. In embodiments of the invention, single-stranded gRNA (sgRNA) is used as part of the RNP complex of the invention.

[0052] Any gRNA disclosed herein may be chemically unmodified—that is, utilizing naturally occurring nucleotides. Alternatively, the gRNA may contain one or more modified nucleotides and / or a modified backbone. For example, a modified gRNA may contain one or more 2'-O-methylphosphothioester nucleotides, which may be located at the 5' end, the 3' end, or both. Alternatively, the gRNA may contain at least one non-naturally occurring nucleotide, such as any nucleotide described in US-2020 / 0224234-A1.

[0053] Suitable for inclusion in the target StPPO2 Exemplary spacer sequences in the gRNA of genes are shown in Table 2 below. In embodiments of the invention, the spacer sequence is located approximately 700 to approximately 1200 nucleotides downstream of the start codon. StPPO2 Antisense strand hybridization (ATG) of the gene. In a further embodiment, the spacer sequence is located approximately 700 to approximately 800 or approximately 1100 to approximately 1200 nucleotides downstream of the start codon (ATG) and... StPPO2Antisense hybridization of genes. Exemplary spacer sequences are particularly suitable for inclusion in gRNA sequences used in Cas12a-based gene editing systems, most typically ErCas12a / MAD7. These exemplary spacer sequences have demonstrated unexpectedly good utility in achieving gene editing events that result in mutations in the genomes of more than one different potato variety. Therefore, unlike previous gene editing strategies, the gRNAs provided herein can be used in multiple heterologous varieties to reduce or eliminate undesirable PPO2-mediated browning reactions in potato tubers. The gRNAs provided herein enable robust, high-efficiency editing across varieties while introducing few or no off-target effects or off-target editing into the potato genome.

[0054] Cas12a RNA-guided endonucleases, such as ErCas12a / MAD7, bind to a genomic region that matches a designed RNA spacer sequence, located immediately upstream of the PAM site. At this location, the MAD7 endonuclease uses a single RuvC-like endonuclease domain to cleave DNA in an interleaved manner, leaving a 4-nucleotide 5'-protrusion at the distal end of the PAM at the genomic target site. The resulting two cleavage sites are located 19 bases after the PAM on the PAM strand (sense strand) and 23 bases on the non-PAM strand (antense strand). Unlike Cas 12a endonucleases (such as MAD7), typical Streptococcus pyogenes (Streptococcus pyogenes)... Streptococcus pyogenes The Cas9 protein uses two nuclease domains (HNH and RuvC-like) to break each DNA strand and generates nearly synchronous blunt-ended DSBs near the PAM. The Cas12a RNA-guided endonuclease enhances the ability of producing sticky ends when cleaving target DNA, thereby promoting the generation of indels and knockout frameshift mutations at the target site. StPPO2 Gene mutations can be used to completely knock out gene function, or can produce allelic variants with reduced or impaired PPO2 activity.

[0055] The site-directed endonuclease system disclosed herein, comprising one or more gRNAs and at least one RNA-guided nuclease (e.g., Cas12a), can be delivered to target cells or protoplasts (e.g., potato cells or protoplasts) using any conventional method for gene editing of target genes. In some embodiments, the components of the nuclease system disclosed herein can be delivered to target cells separately, simultaneously, or sequentially. In other embodiments, the components of the nuclease system can be delivered together to the target, for example, as a complex. In some cases, the gRNA and the RNA-guided endonuclease can be pre-complexed together to form a ribonucleoprotein (RNP), which can be delivered to target cells or protoplasts using conventional techniques. Therefore, the phrase "ribonucleoprotein complex" or "RNP" as used herein refers to a ribonucleoprotein complex having CRISPR-associated endonuclease activity.

[0056] In an embodiment of the present invention, the potato ( S. tuberosum Plants, cells, plant parts, seeds, other propagable material and their offspring can be found in one or more... StPPO2 Mutations in the endogenous alleles of a gene can impair its functionality. StPPO2 Protein expression is reduced or completely inhibited. Therefore, in some cases, detectable levels of polyphenol oxidase activity are significantly reduced or even completely undetectable in plants, cells, plant parts, seeds, other propagable materials, and progeny. In specific embodiments of the invention, the levels of polyphenol oxidase activity in plants, cells, plant parts, seeds, other propagable materials, and their progeny are sufficiently reduced to substantially eliminate polyphenol oxidase-catalyzed browning reactions in tubers. In other embodiments of the invention, when combined with untreated... StPPO2 When compared with tubers of the same variety with intragenetic mutations, the levels of polyphenol oxidase activity in the plant, cells, plant parts, seeds, other propagable material and their progeny were sufficiently reduced to substantially reduce PPO-catalyzed browning reactions (e.g. caused by quinone accumulation) in the tubers by at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 40%, 30% and 20%.

[0057] Potato bruising is an industry-wide concern, leading to the waste of significant quantities of high-quality potatoes. Potato bruising is an enzymatic process similar to that of potato browning, mediated by PPO. In 1994 alone, the total economic loss from potato bruising in the United States reached $298.9 million (Brook, 1996). Current data suggests that potato bruising could have an economic impact of $600 million in the United States alone. Typically, potatoes are stored for about a year after harvest and are frequently subjected to stress bruising due to storage conditions and handling during harvesting and transportation. Bruised potatoes are often discarded, resulting in reduced market yields. The potato according to the present invention (… S. tuberosum Plants, cells, plant parts, seeds, other propagable materials, and their progeny can exhibit significantly reduced or even completely undetectable levels of polyphenol oxidase activity. Therefore, the plants, cells, plant parts, seeds, other propagable materials, and progeny described in this article can reduce bruising sensitivity, thereby leading to increased market yield.

[0058] According to embodiments of the present invention, a method is provided for the efficient engineering modification and recovery of plants, plant cells, or other propagable plant materials (e.g., seeds, cuttings, callus, protoplasts, or any other tissue cultures of regenerable cells), said method comprising a non-wild-type PPO gene expression pattern, particularly a non-wild-type PPO gene expression pattern. StPPO2 Gene expression. Typically, plants, plant cells, or other propagable plant material originate from Solanum species, more preferably from... Solanum tuberosum Varieties—specifically, potato varieties, of which there are thousands. Potato ( Solanum tuberosumThere are two main subspecies: the Andean subspecies (andigena or Andean); and the tuberosum subspecies (tuberosum or Chilean). Generally speaking, well-known cultivars include, but are not limited to, brown-skinned potatoes, red potatoes, white potatoes, yellow potatoes (also known as Yukons), and purple potatoes. Common varieties, also known as cultivars, include, but are not limited to: Abbot, Accent, Adirondack Blue, Adirondack Red, Agata, Agria, Almond, Alturas, Amandine, America, 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, Chiloé, Cielo, Ciklamen, Clavela Blanca, Clearwater Russet, Colleen, Congo, Cosmos, Dakota Russett, Désirée, 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, MagicMolly, 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, Pachacoña, Patrones, Pimpernel, PinkEye, 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 (selected line), Russet Norkotah (selected line), 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, YellowFinn, and Yukon Gold. Any or substantially all cultivars can be transformed or modified as disclosed herein. In a particular embodiment of the invention, the potato variety is selected from Russet Burbank and / or Russet Norkotah 278.

[0059] The inventors discovered that, although StPPO2 It is a relatively small gene, but it has a large number of PAM sites recognized by the Cas12a enzyme, such as ErCas12a / MAD7—namely, YTN, YTTN, or YTTV sites, where Y = C or T, N = any nucleobase, and V = any base except T, thus providing a range of potential target sites for gene editing. However, due to StPPO2 Belonging to the broader PPO gene family, many potential target sites are conserved among PPO genes and are likely to lead to off-target genome editing of homologous PPO genes in the potato genome. Furthermore, due to the highly polymorphic alleles in potato varieties, most identified target sites exhibit one or more polymorphic events on potential gRNA binding sequences at DNA recognition sites in multiple potato varieties, which could lead to non-editing of alleles with different sequences. This was confirmed in cross-varietal trials of promising candidate sgRNAs. Therefore, identifying promising sgRNAs usable across multiple varieties requires more than simple trial-and-error screening. In embodiments of the invention, a method for targeting sgRNAs from multiple potato varieties is provided. StPPO2 sgRNAs exhibit high specificity and gene editing efficiency, but show low or no off-target effects.

[0060] In a specific embodiment of the present invention, potato plant cells, tubers, and / or whole plants are provided, which are located at a distance from... StPPO2 At least one allele of the gene contains an insertion or deletion mutation in the 700-1200 base pair region of the ATG start codon. In a specific embodiment of the invention, potato plant cells, tubers, and / or whole plants are provided, which are located at a distance from... StPPO2 At least one allele of the gene contains an insertion or deletion mutation in the 700-800 base pair region of the ATG start codon. In a specific embodiment of the invention, potato plant cells, tubers, and / or whole plants are provided, which are located at a distance from... StPPO2 At least one allele of the gene contains an insertion or deletion mutation in the 1100-1200 base pair region of the ATG start codon. In one specific embodiment of the invention, a potato plant cell, tuber, and / or whole plant is provided, comprising [materials from...]. StPPO2 An insertion or deletion mutation in the 700-800 base pair region and the 1100-1200 base pair region of the ATG start codon of at least one allele of a gene. The described mutation in potato plant cells, tubers, and / or whole plants 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 a 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 cross-strand breaks in DNA. Optionally, the cross-strand breaks in DNA are due to the gene-editing event described herein. In one embodiment of the invention, the mutation is a deletion of less than about 20 nucleotides, less than about 15 nucleotides, less than about 10 nucleotides, and optionally less than about 5 nucleotides.

[0061] In some embodiments, the present invention provides having modified StPPO2 Potato plant cells, tubers, and / or the whole plant containing genes. In some embodiments, the present invention provides [a specific type of potato plant]. StPPO2 The genes of potato plant cells, tubers and / or whole plants, said StPPO2 The gene has 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 sequence having StPPO2 The genes of potato plant cells, tubers and / or whole plants, said StPPO2 The gene has one or more alleles, including SEQ ID NO: 6, SEQ ID NO: 7 and / or SEQ ID NO: 8.

[0062] In some embodiments, the present invention provides potato plant cells, tubers, and / or whole plants that contain polyphenol oxidase 2 (PPO2). StPPO2 Intra-gene distance StPPO2 The ATG start codon of the gene contains at least one allele mutation within the 700-1200 base pair region, wherein the mutation produces a phenotype of reduced or eliminated polyphenol oxidase activity compared to potato plant cells without the mutation. In some embodiments, the mutation is located at a distance from... StPPO2 The mutation occurs within the 700-800 base pair region of the ATG start codon in the gene. In some implementations, the mutation is located at a distance from... StPPO2 The region within 1100-1200 base pairs of the ATG start codon of the gene. In some embodiments, potato plant cells, tubers, and / or the whole plant are in StPPO2 The gene contains mutations in at least two alleles, wherein the mutations are located at a distance from the gene. StPPO2 The regions within 700-800 and 1100-1200 base pairs of the ATG start codon of the gene.

[0063] In a further embodiment, the present invention provides the use of the plants, plant parts, cells, or seeds described herein in agriculture and / or in the production of human and / or animal food. In another embodiment, the present invention provides the use of the plants, plant parts, cells, or seeds described herein in plant breeding methods and / or in the production of hybrid seeds or other propagable materials.

[0064] In embodiments of the present invention, an RNP complex containing the gRNA described herein can be introduced into plant cells or protoplasts using techniques known to those skilled in the art. For example, the RNP complex can be formed in vitro and mixed directly with the recipient protoplast. Alternatively, the plant cells or protoplasts can be transformed with one or more plasmids or viral vectors that express Cas endonucleases and gRNA in the cells.

[0065] The present invention is further illustrated by the following non-limiting embodiments. Example

[0066] Target commercial product selection:

[0067] Based on the potato production area and main uses, four potato varieties were selected for further analysis. StPPO2 Research.

[0068] Russet Burbank —The main products produced in the United States are used in the food processing market, and to a lesser extent, they are also sold in the farmers' market.

[0069] Russet Norkotah —A variety widely grown in the United States, primarily targeting the fresh potato market.

[0070] Atlantic —The most produced potato variety for potato chip processing in the United States.

[0071] Variety “G” – a proprietary variety grown in the United States and Europe for the vegetable market, but known to be highly susceptible to bruising.

[0072] Acquiring germplasm resources:

[0073] The selected microplants were obtained through full cooperation with relevant breeding institutions and by requesting tissue-cultured microplants from the Scottish Agricultural Science Agency. For its applicability, the genetic material was obtained in accordance with the Nagoya Protocol and its country of origin has been confirmed as the United States and the United Kingdom.

[0074] Obtaining genetic material:

[0075] Following the manufacturer's instructions, whole leaves were taken from each potato variety and DNA was extracted using the Zymo Quick DNA Plant / Seed Extraction Kit. For its applicability, the genetic material was obtained in accordance with the requirements of the Nagoya Resolution.

[0076] StPPO2 Sequencing:

[0077] Announced StPPO2 The sequence, derived from Thygesen et al. (Plant Physiol. (1995) 109: 525-531), is available in the GenBank database (https: / / www.ncbi.nlm.nih.gov) under ID U22921.1 (see Table 1) and is located on chromosome 8. This sequence is used to design multiple coverage sequences. StPPO2 Primer pairs for the gene body. Then, PCR was performed using DNA from each variety to amplify the gene. StPPO2 The PCR products were purified and then subjected to NGS amplicon sequencing. The sequencing results were first used to determine how many alleles were amplified in each primer pair by using the number and percentage of detected polymorphisms. Primer pair sequences for amplifying alleles in all four varieties were plotted for U22921.1 to detect conserved regions with low polymorphic variation across all varieties. These regions were then compared between varieties, and selection was made... StPPO2 gRNA design is performed on a region: specifically the 700-1200 base pairs following the ATG start codon of the U22921.1 gene. Table 1 - U22921.1 potato tuber polyphenol oxidase PPO (POT32 allele) mRNA, complete cds [SEQ ID NO: 1]

[0078] Target selection:

[0079] The RGEN tool Cas Designer (http: / / www.rgenome.net) was used to select gene editing targets, with MAD7 selected as the expected nuclease and the potato genome as the target organism. The 700-1200 bp portion of the sequence with ID U22921.1 was used as input to obtain sgRNA options. Potential off-target effects of the sgRNA were analyzed, and only single targets were selected from the potato genome assembly DM_1-3_516_R44_potato_genome_assembly.v6.1. StPPO2 And at most two mismatched sgRNAs were selected for further investigation. A series of five gRNA candidate sequences were chosen, covering the most conserved portion of the identified part of the gene (see...). Figure 1Starting with the alleles of the G (proprietary) potato variety, we determined whether the gRNA would bind to all four alleles, and selected three particularly suitable gRNAs: sgRNA_6, sgRNA_14, and sgRNA_28. The target binding sequences of sgRNA_6, sgRNA_14, and sgRNA_28 are shown in Table 2 below. Table 2 - Potato tuber polyphenol oxidase PPO (POT32 allele) targeting sgRNA [SEQ ID NO:2-4]

[0080] Protoplast isolation and transfection:

[0081] Protoplasts were isolated from young leaves of potato seedlings grown under tissue culture conditions. The leaves were cut into strips and cultured in a medium to promote plasmolysis. Then, an enzyme solution was used instead of the plasmolysis medium to promote cell wall degradation. When the cell wall was fully digested, the solution containing free cells (i.e., protoplasts) was collected and filtered several times to obtain purified protoplasts. The protoplasts were counted and diluted at a specific density in a conductive medium for transfection. Simultaneously, ribonucleoproteins (RNPs) were prepared by mixing predetermined amounts of ErCas12a and sgRNA in a buffer solution for later culture as needed. RNPs were added to 1 volume of diluted protoplasts, followed by 1 volume of PEG solution. The liquid was homogenized, incubated for several minutes, and then washed with a specific medium. The protoplasts were then resuspended at a specific density in regeneration solution A.

[0082] Protoplast regeneration:

[0083] Protoplasts in regeneration solution A were mixed with 1 volume of alginate solution and placed in a calcium-containing medium to promote alginate solidification. The alginate matrix containing protoplasts was cultured in regeneration solution A for several days. At a suitable developmental stage, regeneration solution A was replaced with regeneration solution B. The protoplasts developed into individual calluses, which were released from the alginate matrix and spread on regeneration solution B. After several days of culture, larger calluses were transferred to regeneration medium C, and then to D, to promote shoot growth. The shoots were collected in regeneration medium E to form roots. After several days, fully developed seedlings were obtained.

[0084] gRNA efficiency in plants:

[0085] Three gRNAs, in the form of ribonucleoprotein (RNP) complexes, were transfected into protoplasts of the G (proprietary) variety via PEG-mediated transfection. Samples were collected 7 days later for DNA extraction, PCR amplification, and purification, and then sent for NGS amplicon sequencing. gRNA efficiency was measured as the percentage of edited reads at the target site compared to reads containing the native sequence. sgRNA_14 and sgRNA_28 (SEQ ID NO:3 and 4) exhibited the highest gene-editing efficiency (see [link to gRNA analysis]). Figure 2 Therefore, it was used for gene editing in other test varieties.

[0086] gRNA efficiency among multiple varieties:

[0087] The locations of sgRNA_14 and sgRNA_28 (SEQ ID NO:3 and 4) in potato varieties G, Atlantic (A), Rustet Burbank (RB), and Rustet Norkotah (RN) were compared. The results are as follows: Figure 3 As shown.

[0088] sgRNA_28 (SEQ ID NO: 4) was identified as being located in a conserved region, and no polymorphism was detected in any plant variety. sgRNA_14 (SEQ ID NO: 3) exhibited positional polymorphism, with some varieties showing single nucleotide substitutions (SNPs) in two alleles in Atlantic and Rustet Burbank, while Rustet Norkotah had one SNP in one allele. Protoplasts were isolated and transfected using sgRNA_14 and sgRNA_28 to test the efficiency of gRNAs across different varieties and to test the effect of SNPs in these varieties compared to the G variety. Samples were collected 7 days later for DNA extraction, PCR amplification, and purification, and then sent for NGS amplicon sequencing. Surprisingly, among the SNP-containing alleles, the sgRNA_28 gene editing efficiency was comparable across all four tested varieties (G, RB, A, RN) (see [link to NGS]). Figure 3 Without being bound by theory, sgRNA_14 was observed to have higher levels of gene editing in variety G (proprietary) compared to other varieties, which may be related to the increased number of SNPs present in the target sequence in Russet Burbank (2), Russet Norkotah (1) and Atlantic (2).

[0089] For plants modified with sgRNA_28, reads were obtained using next-generation sequencing (NGS) and compared with wild-type PPO2 alleles to characterize insertions / deletions resulting from gene editing. Figure 4As shown, referring to the wild-type allele (SEQ ID NO: 5), the intercalary sequence position is highlighted. Comparison with three modifying alleles (SEQ ID NO: 6-8) shows deletions of 4, 10, and 8 nucleotides, respectively. Based on NGS read frequency mapping, this single plant has two alleles with deletions of 4 nucleotides each (SEQ ID NO: 6), and two more alleles with deletions of 8 and 10 nucleotides, respectively (SEQ ID NO: 7 and 8).

[0090] Phenotypic confirmation of traits:

[0091] Fully grown potato plants regenerated from tissue culture are transplanted into greenhouse soil to produce tubers (see...). Figure 5 ).

[0092] Tubers were collected and sectioned, and the tissues were exposed to oxygen and monitored regularly over 48 hours. The color of the tubers was visually assessed at set time points to determine their comparison with the unmodified control variety. StPPO2 Whether gene editing is sufficient to significantly reduce or eliminate oxidative browning.

[0093] Screening for browning phenotypes in potato tubers from original and improved varieties:

[0094] In order to evaluate StPPO2 Knockout of the effect on tuber browning allows for the production of fully developed tuber varieties using potato varieties "G", Atlantic, and Russet Burbank (original varieties). StPPO2 Knockout gene-edited lines (improved varieties). Both groups of plants were grown in a greenhouse, and tubers were harvested for browning analysis.

[0095] Mechanically induced browning was induced by maximizing cell damage through peeling and grinding of the tubers. Images were taken over 14 hours to monitor the browning process. By the end of this period, the original varieties exhibited a nearly black color due to melanin production, while browning in the improved varieties was negligible. These results confirm that all four tested varieties... StPPO2 Allele mutations can effectively eliminate browning reactions caused by damage. Screening results are as follows: Figure 6A -C is shown.

[0096] Comparison of the growth of original and improved plant varieties in a greenhouse:

[0097] In order to evaluate StPPO2 Whether gene editing and protoplast regeneration of whole plants affect plant growth and yield was investigated by planting the original variety "G" and the improved variety (knockout) side by side in a greenhouse. StPPO2 Before transplanting the two groups of plants into the soil of the greenhouse environment, they were cultured in tissue culture medium for 30 days.

[0098] Forty-eight plants were planted for each strain, distributed across four trays, with 12 plants per tray. After 45 days, aboveground biomass and tuber weight were collected and compared between the original strain "G" and the improved strain. Data are displayed in bar graphs to compare characteristics between strains. Figure 7A and 7B The results showed no significant differences between the original and improved lines, indicating that the protoplasts... StPPO2 Knockout and regeneration do not have a negative impact on plant development or yield.

[0099] Potato tubers of original and improved varieties StPPO2 Enzyme activity:

[0100] In addition to visual assessment of browning, the following were also assessed: StPPO2 Enzyme activity was assessed. Tubers from the original and improved strains were randomly selected, and cylindrical samples were extracted using a punch. Sections (2 mm) were incubated in a buffer solution containing L-DOPA (a substrate of PPO2). Control sections were incubated in a buffer solution without L-DOPA to subtract background signal. After 12 minutes, the enzyme activity was indirectly assessed by comparing the color change of the buffer solution caused by the conversion of L-DOPA to melanin. StPPO2 Enzyme activity was measured. Images of the buffer solution for each sample were taken, and pixel intensity was quantified using ImageJ software. Background signal was subtracted to determine relative intensity. Color changes were compared between the original and improved lines. Results showed that enzyme activity was significantly reduced in the improved lines compared to the original lines. The results are shown in Figure 8. Specifically, the activity of the improved line of variety "G" was reduced by 2-3 times (…). Figure 8A ), and improved Atlantic Figure 8B ) and Russet Burbank ( Figure 8C The activity of the strain decreased by 10-15 times. These results indicate that by knocking out... StPPO2 The ability to produce melanin from L-DOPA was eliminated, demonstrating the non-browning phenotype of the tubers.

[0101] In the original strain and the improved strain containing in-frame mutations, the tubers of variety "G" potato... StPPO2 Enzyme activity:

[0102] In the improved strains of variety "G" StPPO2 Enzyme activity was analyzed in this improved strain, which contained one in-frame mutation and three frameshift mutations. These strains exhibited activity levels comparable to those with four frameshift mutations, indicating that sg28 (i.e., sgRNA_28) induces enzyme activity against enzymes containing one in-frame mutation and three frameshift mutations. StPPO2 The loss of key amino acids required for mediated oxidation. The results are as follows: Figure 9 As shown.

[0103] PPO activity can also be determined enzymatically by measuring the initial oxygen uptake rate of homogenized tissue extracts in a 50 mM sodium phosphate (pH 6.0) solution at 25°C. The reaction is initiated by adding the substrate 4-methylcatechol to a final concentration of 2 mM. One unit of activity is defined as the amount of enzyme that catalyzes the consumption of 1 pmol of oxygen per minute under the assay conditions.

[0104] Gene editing efficiency of sg28 in different potato varieties:

[0105] In order to achieve StPPO2 Gene editing was performed using the CRISPR-Cas system. Cas12 binding sites were identified in the target gene, with a preference for the central region, where nucleotide insertion or deletion is most effective for gene knockout. Multiple guide RNAs were evaluated in vitro and in vivo. Among them, sg28 (i.e., sgRNA_28) showed the highest activity and was further tested across different potato varieties.

[0106] To evaluate the editing of sg28 in protoplasts of different potato varieties StPPO2 To assess gene editing efficiency, the sg28 guide RNA binds to ErCas12 to form a ribonucleoprotein (RNP) complex. This complex was transfected into protoplasts of five potato varieties: variety "G", Atlantic, Russet Burbank, Russet Norkotah, and Ranger Russet. After transfection, the protoplasts were embedded in a matrix solution and incubated for 7 days. DNA was extracted, the target region was amplified by PCR, and amplicon sequencing was performed. Editing efficiency was calculated using CRISPRESSO based on next-generation sequencing (NGS) data. Multiple biological replicate analyses were performed for each variety. The results showed that sg28 achieved gene editing efficiencies exceeding 30% in all tested varieties. (See attached figures.) Figure 10 As shown.

[0107] While specific embodiments of the invention have been described in detail herein, they are merely illustrative. The above embodiments are not intended to limit the scope of the appended claims. The selection of the type of nucleic acid starting material, target clone, or library used is considered conventional matter that can be grasped by those skilled in the art with knowledge of the embodiments described herein. The inventors anticipate that various substitutions, alterations, and modifications can be made to the invention without departing from the spirit and scope of the invention as defined by the claims.

Claims

1. A potato plant cell containing polyphenol oxidase 2 ( StPPO2 A mutation in at least one allele of a gene, said mutation in StPPO2 The mutation occurs within the 700-1200 base pair region of the ATG start codon of the gene, where, compared to potato plant cells without the mutation, the mutation produces a phenotype of reduced or eliminated polyphenol oxidase activity.

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

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

4. The plant cell according to claim 1, wherein the mutation is contained in StPPO2 Within multiple alleles of a gene.

5. The plant cell according to claim 4, wherein the mutation is contained in the distance from StPPO2 The region of 700-800 base pairs and the region of 1100-1200 base pairs of the ATG start codon of the gene.

6. The plant cell according to any one of claims 1-5, wherein... StPPO2 The gene contains one or more alleles, which contain the sequence shown in SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO:

8.

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

8. A potato tuber comprising the plant cells of any one of claims 1-7.

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

10. The potato tuber of claim 9, wherein the tuber exhibits reduced PPO-mediated browning reaction compared to non-GMO potato tubers of the same variety or species.

11. The potato tuber according to any one of claims 8-10, wherein the tuber is derived from a potato variety selected from the following: Abbot, Accent, Adirondack Blue, Adirondack Red, Agata, Agria, Almond, Alturas, Amandine, America, 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, CaribouRussett, Cabritas, Camota, Cardinal, Centennial Russet, Challanger, Charlotte, Chelina, Chieftain, Chiloé, Cielo, Ciklamen, Clavela Blanca, Clearwater Russet, Colleen, Congo, Cosmos, Dakota Russett, Désirée, 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, LadyFelicia, Lady Rosetta, Lamoka, Laura, Linda, Magic Molly, Marfona, Marilynn, MarisBard, Maris Piper, Maris Peer, Marquis, Mellody, MeruMix, Moonlight, Mozart, Mukorino, Nadine, Natascha, Nectar, Ngure, Nicola, Norland, Novello, Nyayo Mukori, Orla, Pachacoña, Patrones, Pimpernel, Pink Eye, Pink Fir Apple, Pinto Gold, Pirol, Primura, Ratte, Picasso, Purple Majesty, Purple Viking, Purplu, Ranger Russet, RedGold, 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 (selected line), Russet Norkotah (selected line), 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.

12. A potato plant or a plant part thereof comprising plant cells according to any one of claims 1-7, wherein the plant exhibits a phenotype of reduced browning response mediated by PPO compared with a non-transgenic potato plant.

13. The plant of claim 12, wherein the plant part is selected from leaves, pollen, ovules, fruits, rhizomes, young branches, tubers, flowers, and cells.

14. A tissue culture of regenerative cells of the plant or a plant part thereof as described in claim 12 or 13.

15. A Cas12a ribonucleoprotein (RNP) complex comprising a Cas12a endonuclease and a guide RNA (gRNA), wherein the guide RNA is associated with a potato variety's polyphenol oxidase 2 (… StPPO2 Hybridization with target sequences contained within the gene.

16. The RNP complex of claim 15, wherein the gRNA is contained in... StPPO2 Hybridization of target sequences within a region of a gene, wherein the region is defined as StPPO2 The region 700-1200 base pairs downstream of the ATG start codon in a gene.

17. The RNP complex of claim 16, wherein the gRNA comprises a sequence selected from SEQ ID NO: 2-4.

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

4.

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

20. The RNP complex according to any one of claims 15-19, wherein the gRNA is contained in at least two potato varieties. StPPO2 Hybridization of target sequences within genes. 21.On the 15-20th day of the month of the 15-20th century, one of the most expensive snowflakes in the RNP is the Abbot, Accent, and Adirondack Blue、Adirondack Red、Agatha、Sour、Almond、Heights、Amandine、Americar、Amin、Annabelle、Anya、Arran Victory、Atlantic、Arizona、AustrianCrescent、Bamberg、Belana、Belle de Fontenay, BF-15, Bildtstar, Bintje, Blackberry, Blue Congo, Blue Danube, Bonnotte, Cinnamon Russet, Cara, Caribou Russett, Cabritas, Camota, Cardinal, Centennial Russet、Challanger、Charlotte、Chelina、Chieftain、Chiloé、Cielo、Cyklamen、White Carnation、Clearwater Russet、Colleen、Congo、Cosmos、Dakota Russett、Désiré、Draga、Duke of York、Edzell Blue、Elba、Faraja、Feldeslohn、Fianna、Fingerling、Flava、Fontana、Frisia、Gala、Georgina、German Butterball、GoldRush、Golden Wonder、Golf、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, Mary Bard, Mary Piper, Mary Peer, Marquis, Mellody, MeruMix, Moonlight, Mozart, Mukorino, Nadine, Natascha, Nectar, Ngure, Nicola, Norland, Novello, Nyayo Mukori, Orla, Pachacoña, Patrones, Pimpernel, PinkEye, Pink Fir Apple, Pinto Gold, Pirol, Primura, Ratte, Picasso, Purple Majesty, Purple Viking, Purplu, Ranger Russet, Red Gold, Red Norland, Red Pontiac, RedRascal, Red Thumb, Reveille Russet, Robijn, Robinta, Rockstar, Romano, Rooster, Roslin Chania, Roslin Tana, Roslin, Royal, Rudolph, Rua, Rugano, Russet Burbank (selected line), Russet Norkotah (selected line), 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.

22. The plant cell according to claim 7, wherein the cross-strand breaks in the DNA are the result of gene editing using the RNP complex according to any one of claims 15-21.

23. An isolated guide RNA comprising a nucleotide sequence selected from any one of SEQ ID NO: 2-4.

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

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

26. A method for reducing or eliminating PPO activity in plant cells from a potato species, the method comprising targeting the plant cell genome... StPPO2 One or more alleles of a gene are mutated, wherein the mutation is caused by a gene located at... StPPO2 The frameshift mutation is caused by an insertion or deletion in the 700-1200 base pair region of the ATG start codon, and 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, caused by a mutation located at... StPPO2 It is caused by an insertion or deletion within the 700-800 base pair region of the ATG start codon of a gene.

28. The method of claim 26, wherein the mutation is a frameshift mutation, caused by a mutation located at... StPPO2 It is caused by an insertion or deletion within the 1100-1200 base pair region of the ATG start codon of a gene.

29. The method of claim 26, wherein the mutation is a frameshift mutation, caused by a mutation located at... StPPO2 Insertion or deletion within the 700-800 base pair region of the ATG start codon of a gene, as well as insertion or deletion within the 1100-1200 base pair region, can cause this.

30. The method according to any one of claims 26-29, wherein the CRISPR / Cas gene editing event is the result of a CRISPR / Cas12a directed gene editing event performed using the RNP according to any one of claims 15-21.

31. The method according to any one of claims 26-30, wherein the method comprises regenerating an intact plant from the plant cells, wherein the intact plant exhibits a phenotype of reduced or eliminated PPO activity compared to a potato plant without the mutation.

Images