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Living Modified Organism
(LMO)
The image below identifies the LMO through its unique identifier, trade name and a link to this page of the BCH. Click on it to download a larger image on your computer. For help on how to use it go to the LMO quick-links page.
Late blight resistant potato
EN
Potato line (Solanum tuberosum cv. Yulia) edited at the StDMR6-1 and StCHL1 loci using CRISPR/Cas9
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Person:DSc Oksana Yurievna UrbanovichHead of the Laboratory, Laboratory of Molecular GeneticsPhone: +375 17 379 91 80,Fax:Email: O.Urbanovich@igc.by,Website:Related OrganizationSSI "Institute of Genetics and Cytology at NAS Belarus" ()Academic or research instituteAkademicheskaya, 27Minsk,
220072, BelarusPhone: +375 17 379 91 80,Fax:Email: O.Urbanovich@igc.by,Website:
The modified potato (Solanum tuberosum, cv. Yulia) contains knockout mutations in the endogenous susceptibility genes StDMR6-1 and StCHL1 generated using CRISPR/Cas9-mediated genome editing. These genes are involved in plant immune regulation and salicylic acid metabolism. Their disruption impairs susceptibility pathways and is expected to enhance resistance to late blight caused by Phytophthora infestans. The transformation was carried out using Agrobacterium tumefaciens-mediated transfer of a T-DNA construct encoding the Streptococcus pyogenes Cas9 nuclease, guide RNAs targeting StDMR6-1 and StCHL1, and a hygromycin resistance selectable marker. Foreign DNA sequences, including the selectable marker, are reported to remain integrated in the genome of the modified line.
The term “Recipient organism” refers to an organism (either already modified or non-modified) that was subjected to genetic modification, whereas “Parental organisms” refers to those that were involved in cross breeding or cell fusion.
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BCH-ORGA-SCBD-12106-6 Organism Solanum tuberosum (Potato, SOLTU)Crops
The Yulia potato is the variety of Belarusian selection. It was included in the State Register of Varieties of the Republic of Belarus in 2021.
EN
binary vector pRGEB31
EN
- Agrobacterium-mediated DNA transfer
- Gene editing (e.g. CRISPR-Cas, etc.)
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0.381 kb
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0.000 kb
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0.000 kb
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0.076 kb
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0.346 kb
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4.101 kb
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0.253 kb
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0.175 kb
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1.026 kb
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0.678 kb
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Some of these genetic elements may be present as fragments or truncated forms. Please see notes below, where applicable.
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BCH-GENE-SCBD-14991-8 Hygromycin B phosphotransferase gene | Escherichia coli (ECOLX)Protein coding sequence | Resistance to antibiotics (Hygromycin),Selectable marker genes and reporter genes
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BCH-GENE-SCBD-277306-1 CRISPR-associated endonuclease 9 | Streptococcus pyogenes (STRPY)Protein coding sequence | Genome editing
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BCH-GENE-SCBD-100269-8 Nopaline Synthase Gene Terminator | Agrobacterium tumefaciens (Agrobacterium)Terminator
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BCH-GENE-SCBD-100287-7 CaMV 35S promoter | Cauliflower mosaic virus (CaMV)Promoter
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BCH-GENE-SCBD-299208-1 Downy Mildew Resistance 6 | Solanum tuberosum (Potato, SOLTU)Protein coding sequence | Resistance to diseases and pests
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BCH-GENE-SCBD-299209-1 RNA Polymerase III promoter | Oryza sativa (Rice, ORYSA)Promoter
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BCH-GENE-SCBD-277309-1 Synthetic cas9 guide RNAGuide RNA
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BCH-GENE-SCBD-299214-1 StCHL1 | Solanum tuberosum (Potato, SOLTU)Protein coding sequence | Resistance to diseases and pests
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BCH-GENE-SCBD-100290-6 CaMV 35S terminator | Cauliflower mosaic virus (CaMV)Terminator
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BCH-GENE-SCBD-299491-1 Synthetic DMR6 guide RNAGuide RNA
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BCH-GENE-SCBD-299490-1 Synthetic CHL1 guide RNAGuide RNA
The modified potato contains three cassettes for Streptococcus pyogenes CRISPR-associated endonuclease 9 (cas9), guide RNAs targeting the endogenous potato genes StDMR6-1 and StCHL1, and Escherichia coli hygromycin phosphotransferase (hyg).
-The guide RNA expression cassette contains guide RNAs complementary to protospacer sequences in the endogenous potato genes StDMR6-1 and StCHL1 fused to a guide RNA scaffold. Expression of the guide RNAs is regulated by the Oryza sativa OsU3 RNA polymerase III promoter and a small (6pb) RNA transcription terminator (pol A III terminator).
-The cas9 coding sequence is under the control of the Cauliflower mosaic virus 35S promoter and the Agrobacterium tumefaciens nopaline synthase (NOS) terminator.
-The hyg coding sequence is used as a selectable marker during plant transformation and it is under the control of the Cauliflower mosaic virus 35S promoter and terminator.
Note:
• According to information provided by the developer, foreign DNA sequences remain integrated in the genome of the modified line, including the hygromycin resistance marker gene.
• The pRGEB31 transformation vector additionally contains a kanamycin resistance gene used for bacterial selection and other cloning elements; however, information was not provided regarding the integration of vector backbone sequences into the potato genome.
• The binary vector pRGEB31 is not self-transmissible and requires the helper plasmid pRK2013 for transfer between bacterial cells. The helper plasmid was not present in the Agrobacterium tumefaciens strain used for transformation and is absent from the transformed potato genome.
-The guide RNA expression cassette contains guide RNAs complementary to protospacer sequences in the endogenous potato genes StDMR6-1 and StCHL1 fused to a guide RNA scaffold. Expression of the guide RNAs is regulated by the Oryza sativa OsU3 RNA polymerase III promoter and a small (6pb) RNA transcription terminator (pol A III terminator).
-The cas9 coding sequence is under the control of the Cauliflower mosaic virus 35S promoter and the Agrobacterium tumefaciens nopaline synthase (NOS) terminator.
-The hyg coding sequence is used as a selectable marker during plant transformation and it is under the control of the Cauliflower mosaic virus 35S promoter and terminator.
Note:
• According to information provided by the developer, foreign DNA sequences remain integrated in the genome of the modified line, including the hygromycin resistance marker gene.
• The pRGEB31 transformation vector additionally contains a kanamycin resistance gene used for bacterial selection and other cloning elements; however, information was not provided regarding the integration of vector backbone sequences into the potato genome.
• The binary vector pRGEB31 is not self-transmissible and requires the helper plasmid pRK2013 for transfer between bacterial cells. The helper plasmid was not present in the Agrobacterium tumefaciens strain used for transformation and is absent from the transformed potato genome.
CRISPR/Cas9-mediated genome editing introduced knockout mutations in the endogenous potato genes StDMR6-1 and StCHL1. These mutations are expected to result in loss of function of the corresponding proteins, which are associated with suppression of plant immune responses. Disruption of these susceptibility genes enhances resistance to late blight caused by Phytophthora infestans.
- Research
Detection is performed by conventional PCR targeting sequences within the CRISPR/Cas9 expression cassette. The method uses primer pairs U3-F/35-R and 35-f/35-R to amplify fragments of approximately 1398 bp and 346 bp, respectively, from the integrated construct. PCR products are visualized by agarose gel electrophoresis. Primer sequences and reaction conditions are provided in the risk assessment dossier submitted by the developer.
EN
- Creation of vector constructs carrying the CRISPR/Cas9 system for directed mutagenesis of CHL1 and DMR6-1 Solanum tuberosum genes ( CRISPR/Cas9, solanum, directed mutagenesis ) [ Russian ]
- pRGEB31 binary vector map [ English ]
| Record type | Field | Record(s) | |
|---|---|---|---|
| Country's Decision or any other Communication | LMO identification | 1 | |
| Risk Assessment generated by a regulatory process | Living modified organism(s) | 1 | |
