| Title: | CRISPR/Cas9-mediated fine-tuning of miRNA expression in tetraploid potato |
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| Authors: | ID Lukan, Tjaša (Author)
ID Veillet, Florian (Author)
ID Križnik, Maja (Author)
ID Coll Rius, Anna (Author)
ID Mahkovec Povalej, Tjaša (Author)
ID Pogačar, Karmen (Author)
ID Stare, Katja (Author)
ID Chauvin, Laura (Author)
ID Chauvin, Jean-Eric (Author)
ID Gruden, Kristina (Author) |
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| Files: |  URL - Source URL, visit https://academic.oup.com/hr/article/doi/10.1093/hr/uhac147/6623516
 PDF - Presentation file, download (2,94 MB)
MD5: 2AF8FACAE0FBECAECB7F6677F19FEAD0
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| Language: | English |
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| Typology: | 1.01 - Original Scientific Article |
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| Organization: |  NIB - National Institute of Biology
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| Abstract: | MicroRNAs (miRNAs) are small noncoding RNAs, which modulate the abundance and spatiotemporal accumulation of target mRNAs at transcriptional and post-transcriptional levels and through that play important roles in several biological processes in plants. Here we show that in polyploid species, CRISPR/Cas9 system can be used for fine-tuning of miRNA expression, which can have broader range of applications compared to knock-out mutants. We established the complete pipeline for CRISPR-Cas9-mediated modulation of miRNA expression in potato. It consists of (1) design and assembly of dual sgRNA CRISPR/Cas9 constructs, (2) transient transfection of protoplasts following fast and efficient screening by high resolution melting analysis to select functional sgRNAs, and (3) stable transformation of potato explants with functional sgRNAs and selection of regenerated transgenic lines with desired mutations and desired miRNA abundance based on sequencing and RT-qPCR. We show that miRNA-editing using dual sgRNA approach results in different types of mutations among transgenic lines but also in different alleles of the same plant, which are target site-dependent. The most frequent were short deletions, but we also detected 1-nt insertions (T or G), deletions between two sgRNAs and larger deletions. miRNA abundance correlates with the frequency and type of introduced mutations, as more extensive mutations in more alleles result in lower miRNA abundance. Interestingly, some mutated loci can generate alternative miRNAs, now novel targets were however predicted for those. In all transgenic lines with Cas9 expression, we detected mutations, suggesting high efficiency of Cas9-editing. We confirmed the miRNA-editing efficiency of our optimised approach in two different potato genotypes and three different loci. |
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| Keywords: | CRISPR/Cas9, hypersensitive response (HR)-conferred resistance, immune signaling, live cell imaging, Solanum tuberosum (potato), spatiotemporal analysis, stromules, virus resistance |
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| Publication status: | Published |
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| Publication version: | Version of Record |
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| Publication date: | 30.06.2022 |
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| Year of publishing: | 2022 |
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| Number of pages: | str. 1-15 |
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| Numbering: | Vol. 9 |
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| PID: | 20.500.12556/DiRROS-19442  |
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| UDC: | 57 |
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| ISSN on article: | 2052-7276 |
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| DOI: | 10.1093/hr/uhac147 |
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| COBISS.SI-ID: | 121833731 |
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| Note: | Nasl. z nasl. zaslona;
Opis vira z dne 19. 9. 2022;
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| Publication date in DiRROS: | 17.07.2024 |
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| Views: | 137 |
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| Downloads: | 139 |
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