Abstract
CRISPR/Cas9 technology has become the most efficient method for genome editing in many plant species, including important industrial crops such as potatoes. This study used three target regions (T1, T2, and T3) in gbss exon I, whose sequences were first inserted into the BbsI sites in the appropriate guide RNA (gRNA) vector (pEn-Chimera, pMR203, pMR204, and pMR205), and then localized between the AtU6 promoter and the gRNA scaffold sequence. Expression vectors were constructed by introducing gRNA genes into the pMR287 (pYUCas9Plus) plasmids using the MultiSite Gateway system by attR and attL sites. The three target regions of mutant potato lines were analyzed. The use of CRISPR/Cas9-mediated multiple guide RNA-targeted mutagenesis allowed tri- or tetra-allelic mutant potato lines to be generated. Multiple nucleotide substitutions and indels within and around the three target sites caused a frameshift mutation that led to a premature stop codon, resulting in the production of gbss-knockout plants. Mutation frequencies and analysis of mutation patterns suggested that the stably transformed Cas9/multiple guide RNA expression constructs used in this study can induce targeted mutations efficiently in the potato genome. Full knockout of the gbss gene was analyzed by CAPS, Sanger sequencing and iodine staining. The present study demonstrated successful CRISPR/Cas9-mediated multiple guide RNA-targeted mutagenesis in the potato gbss gene by Agrobacterium-mediated transformation, resulting in an amylose-free phenotype.
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Data availability statement
The sequences of clones generated and analyzed for this study were uploaded into GenBank database of NCBI. All the Row data of the sequences and accession numbers are available in Supplementary Table 4.
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Acknowledgements
We are grateful to the Ministry of Education and Science of the Republic of Kazakhstan for financial support and Prof. Seiichi Toki, National Agriculture and Food Research Organization, for providing vector and teaching the technology.
Funding
This work is supported by grants from the Ministry of Education and Science of the Republic of Kazakhstan [grant number: AP05130386 and AP09259964].
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MS and RY conceived the experiments. LA, KB, and AA conducted the experiments. MS, LA, and TD analyzed the results. MS drafted the manuscript, MS and TD contributed to the final editing of the manuscript. All authors read and approved the final manuscript.
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11248_2023_356_MOESM1_ESM.xlsx
Supplementary Table 2 Effect of acetosyringone and surfactant Silwet L-77 on Agrobacterium-mediated 238 transformation. Direct shoot regeneration of local potato cultivars Astanalyk, Aksor and Tokhtar from 239 internode and leaf explants. Each value represents mean SD of three replicated experiment (xlsx 35 kb)
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Supplementary Table 3 Data including the total number of samples, transformed explants, regenerated explants and regenerated mutated plants (xlsx 65 kb)
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Supplementary Table 4 The Sequences of Clones unloaded into NCBI (analyzed sections of the sequences that inserted to the Fig 8 were highlighted with red T1, blue T2, and green T3 colors). (docx 23 kb)
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Abeuova, L., Kali, B., Tussipkan, D. et al. CRISPR/Cas9-mediated multiple guide RNA-targeted mutagenesis in the potato. Transgenic Res 32, 383–397 (2023). https://doi.org/10.1007/s11248-023-00356-8
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DOI: https://doi.org/10.1007/s11248-023-00356-8