20.500.12556/DiRROS-19442 CRISPR/Cas9-mediated fine-tuning of miRNA expression in tetraploid potato 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. CRISPR/Cas9 hypersensitive response (HR)-conferred resistance immune signaling live cell imaging Solanum tuberosum (potato) spatiotemporal analysis stromules virus resistance CRISPR/Cas9 small RNA rearrangement potato polyploids true false true Angleški jezik Slovenski jezik Neznano 2024-07-17 11:18:42 2024-07-17 11:18:42 2024-07-18 03:54:07 0000-00-00 00:00:00 2022 0 0 Nasl. z nasl. zaslona; Opis vira z dne 19. 9. 2022; str. 1-15 Vol. 9 2022 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 2022-06-30 57 2052-7276 10.1093/hr/uhac147 121833731 https://academic.oup.com/hr/article/doi/10.1093/hr/uhac147/6623516 1 d0327732-440c-11ef-b487-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=26830 RAZ_Lukan_Tjasa_2022.pdf RAZ_Lukan_Tjasa_2022.pdf 1 2AF8FACAE0FBECAECB7F6677F19FEAD0 3d6fe402a834c2b959fbda3b1186e222dd320903ca33ab35b4e5b41dde5b6eee 0ae1b85a-440d-11ef-b487-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=26831 Nacionalni inštitut za biologijo 0 0 0