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Article|30 Jun 2022|OPEN
CRISPR/Cas9-mediated fine-tuning of miRNA expression in tetraploid potato
Tjasa Lukan1 , , Florian Veillet2 , Maja Kriznik1 , Anna Coll1 , Tjasa Mahkovec Povalej1 , Karmen Pogacar1 , Katja Stare1 , Laura Chauvin2 , Jean-Eric Chauvin2 and Kristina Gruden,1
1Department of Biotechnology and Systems Biology, National Institute of Biology, Vecna pot 111, Ljubljana, 1000 Slovenia
2IGEPP, INRAE, Institut Agro, Universite´ de Rennes, Ploudaniel 29260, France
*Corresponding author. E-mail: tjasa.lukan@nib.si

Horticulture Research 9,
Article number: uhac147 (2022)
doi: https://doi.org/10.1093/hr/uhac147
Views: 109

Received: 19 Mar 2022
Accepted: 27 Jun 2022
Published online: 30 Jun 2022

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.