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WO2025149596A1 - Solanum tuberosum à activité réduite de la polyphénol-oxydase - Google Patents

Solanum tuberosum à activité réduite de la polyphénol-oxydase

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Publication number
WO2025149596A1
WO2025149596A1 PCT/EP2025/050489 EP2025050489W WO2025149596A1 WO 2025149596 A1 WO2025149596 A1 WO 2025149596A1 EP 2025050489 W EP2025050489 W EP 2025050489W WO 2025149596 A1 WO2025149596 A1 WO 2025149596A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
stppo2
plant
mutation
russet
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/050489
Other languages
English (en)
Inventor
Luis Andrade
Jovaras KRASAUSKAS
William PELTON
Nicolas KRAL
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Phytoform Labs Ltd
Original Assignee
Phytoform Labs Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Phytoform Labs Ltd filed Critical Phytoform Labs Ltd
Publication of WO2025149596A1 publication Critical patent/WO2025149596A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine

Definitions

  • the invention relates to improvements in reduction of tuber browning in potato (Solatium tuberosum) varieties utilising endonuclease-directed gene editing technologies.
  • 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.
  • a Solanum tuberosum plant cell 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.
  • 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.
  • Figure 5 is a photograph showing gene edited potato plants that were regenerated from tissue culture.
  • 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.
  • the crRNA forms a duplex that binds to the endonuclease, such that the guide RNA and endonuclease form a complex.
  • 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.
  • each gRNA is designed to include a spacer sequence complementary to its genomic target sequence.
  • guide RNA gRNA
  • sgRNA single guide RNA
  • 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).
  • 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).
  • 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.
  • the target sequence is in the region of the StPPO2 gene 700-800 base pairs from the start codon (ATG) of the StPPO2 gene.
  • the gRNA disclosed herein may target any sequence of interest via the spacer sequence comprised within it.
  • 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%.
  • the spacer sequence of the guide RNA and the target sequence in the target gene is 100% complementary.
  • 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.
  • a single gRNA gRNA
  • sgRNA single gRNA
  • any of the gRNAs disclosed herein may be chemically unmodified - i.e. utilising naturally occurring nucleotides.
  • the gRNAs may contain one or more modified nucleotides and/or modified backbones.
  • 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.
  • the gRNAs may comprise at least one non-naturally occurring nucleotide such as any one of those described in US-2020/0224234-A1 .
  • Cas12a RNA guided endonucleases such as ErCas12a/MAD7
  • 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.
  • 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).
  • 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.
  • the plants, cells, plant parts, seeds, other propagatable material and progeny exhibit substantially reduced or even entirely eliminated detectable levels of polyphenol oxidase activity.
  • 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.
  • 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.
  • 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.
  • 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, Schmtstar, 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the staggered double strand break in the DNA is due to a gene editing event as described herein.
  • 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.
  • 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.
  • 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.
  • the mutation is within a region 700-800 base pairs from the ATG start codon of the StPPO2 gene.
  • the mutation is within a region 1100-1200 base pairs from the ATG start codon of the StPPO2 gene.
  • 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.
  • 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.
  • 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.
  • Russet Norkotah -A variety grown on large number of acres in the United States, mainly used in the fresh potato market.
  • 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.
  • 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.
  • 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
  • sgRNA_14 and sgRNA_28 were 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).
  • 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.
  • 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.

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  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
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  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
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Abstract

L'invention concerne des améliorations de la réduction du brunissement des tubercules dans les variétés de pommes de terre (Solanum tuberosum) utilisant des techniques d'édition génique dirigée par endonucléase. Plus particulièrement, l'invention concerne une cellule végétale de Solanum tuberosum comportant une mutation qui génère un phénotype d'activité de polyphénol oxydase réduite ou supprimée par comparaison avec une cellule végétale de Solanum tuberosum dépourvue de la mutation. L'invention concerne également des complexes ribonucléoprotéiques contenant une endonucléase et un ARN guide qui s'hybride avec une séquence cible comportant un gène de polyphénol oxydase 2 (StPPO2) d'une variété de Solanum tuberosum, ainsi que des procédés d'utilisation de ces complexes.
PCT/EP2025/050489 2024-01-09 2025-01-09 Solanum tuberosum à activité réduite de la polyphénol-oxydase Pending WO2025149596A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB2400313.9A GB202400313D0 (en) 2024-01-09 2024-01-09 Solanum tuberosum with reduced polyphenol oxidase activity
GB2400313.9 2024-01-09

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Publication Number Publication Date
WO2025149596A1 true WO2025149596A1 (fr) 2025-07-17

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008046189A1 (fr) * 2006-10-11 2008-04-24 Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of The Province Of Nova Scotia, The Nova Scotia Agricultural College (Nsac) Protéines mises en jeu dans le noircissement des pommes de terre après cuisson
WO2022114692A1 (fr) * 2020-11-25 2022-06-02 주식회사 툴젠 Procédé de production d'un plant de pomme de terre à brunissement supprimé à l'aide d'un système crispr/cas9

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008046189A1 (fr) * 2006-10-11 2008-04-24 Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of The Province Of Nova Scotia, The Nova Scotia Agricultural College (Nsac) Protéines mises en jeu dans le noircissement des pommes de terre après cuisson
WO2022114692A1 (fr) * 2020-11-25 2022-06-02 주식회사 툴젠 Procédé de production d'un plant de pomme de terre à brunissement supprimé à l'aide d'un système crispr/cas9

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GONZ�LEZ MAT�AS NICOL�S ET AL: "Comparative potato genome editing: Agrobacterium tumefaciens-mediated transformation and protoplasts transfection delivery of CRISPR/Cas9 components directed to StPPO2 gene", PLANT CELL, TISSUE AND ORGAN CULTURE (PCTOC), SPRINGER NETHERLANDS, DORDRECHT, vol. 145, no. 2, 25 January 2021 (2021-01-25), pages 291 - 305, XP037427773, ISSN: 0167-6857, [retrieved on 20210125], DOI: 10.1007/S11240-020-02008-9 *
GONZ�LEZ MAT�AS NICOL�S ET AL: "Reduced Enzymatic Browning in Potato Tubers by Specific Editing of a Polyphenol Oxidase Gene via Ribonucleoprotein Complexes Delivery of the CRISPR/Cas9 System", FRONTIERS IN PLANT SCIENCE, vol. 10, 9 January 2020 (2020-01-09), CH, pages 1649 - 1, XP055934576, ISSN: 1664-462X, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/ivip/1664-462X> DOI: 10.3389/fpls.2019.01649 *
TUNCEL AYTUG ET AL: "CRISPR/Cas mediated genome editing in potato: Past achievements and future directions", PLANT SCIENCE, ELSEVIER IRELAND LTD, IE, vol. 325, 26 September 2022 (2022-09-26), pages 1 - 11, XP087219341, ISSN: 0168-9452, [retrieved on 20220926], DOI: 10.1016/J.PLANTSCI.2022.111474 *

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