CRISPR/Cas9-mediated fine-tuning of miRNA expression in tetraploid potato

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.

Recent advances and challenges in potato improvement using CRISPR/Cas genome editing

“…Increased effectiveness of introducing targeted mutations and likelihood of tetra-allelic mutations More complex constructs. More potential off-targets Andersson et al 2017 , Kusano et al 2018 , Carlsen et al 2022 , Lukan et al 2022 Testing plant RNA promoter/-s Arabidopsis U6 promoter ( AtU6 ) Effective and universal for dicotyledonous plants Less effective than native potato promoters Kusano et al 2018 , Banfalvi et al 2020 , Gonzales et al 2021 , Perroud et al 2022 Using geminivirus replicons (GVRs) Elements from the bean yellow dwarf virus (BeYDV) Improved frequency of HR-generated mutations No clear evidence that the GVR-based CRISPR/Cas vectors are more effective in potato than those containing the standard T-DNA backbone Butler et al 2015 , Butler et al 2016 Optimizing CRISPR/Cas delivery method Agrobacterium -mediated stable transformation Possible generation of tetra-allelic mutants. Possible induction of large fragment deletions/insertions Frequently occurring mosaic effects and somaclonal variations in edited plant tissues.…”

“…More than 25% of plants regenerated after stable Agrobacterium -mediated transformation showed the presence of four allelic mutations in the GBSS , whereas the editing effectivity was significantly lower (less than 10% of full allelic mutants) when the vector variants with only two sgRNAs and/or without the dMac3 were used (Kusano et al 2018 ). In the most recently published study, Lukan et al ( 2022 ) demonstrated the effectiveness of using CRISPR/Cas9 to modulate miRNA expression in two potato cultivars, Désirée and Rywal, through the use of dual sgRNA constructs designed for targeting both 5ʹ and 3ʹ ends of three selected MIR genes (Lukan et al 2022 ).…”

“…The techniques for protoplast transfection allow for the temporary activation of CRISPR/Cas elements in target cells. The transient expression system based on transfected protoplasts can be useful to rapidly and efficiently test the functionality of designed CRISPR/Cas constructs (Lukan et al 2022 ) and is frequently used for the generation of transgene-free genome-edited plants (Fig. 1 , left panel) (Zong et al 2018 ; Fossi et al 2019 ; Chauvin et al 2021 ; Nicolia et al 2021 ; Rather et al 2022 ).…”

“…Adaptation of protocols developed for dihaploids or diploid genotypes/species or the cv. Désirée for other potato cultivars often requires additional optimization procedures Andersson et al 2017 , Kusano et al 2018 , Nakayasu et al 2018 , Zheng et al 2021 , Lukan et al 2022 , Carlsen et al 2022 , Toinga-Villafuerte et al 2022 Solutions successfully adapted from other plants Enhancing Cas expression Constitutive promotors: CaMV 35S promoter, duplicated CaMV 35S promoter, ubiquitin 4–2 promoter from parsley High expression of CRISPR/Cas genes. Easy availability of ready-made constructs.…”

“…Possible induction of large fragment deletions/insertions Frequently occurring mosaic effects and somaclonal variations in edited plant tissues. Not recommended for generating non-transgenic genome-edited potatoes Kusano et al 2018 , Veillet et al 2019a , Veillet et al 2019b , Banfalvi et al 2020 , Perroud et al 2022 , Lukan et al 2022 Agrobacterium -mediated transient transformation Possibility to generate non-transgenic genome-edited lines Lower efficiency than in stable transformation. Very low probability to induce large fragment deletion/insertion Veillet et al 2019a , Banfalvi et al 2020 , Perroud et al 2022 Protoplast transfection with plasmids carrying CRISPR/Cas components High frequency of tetra-allelic mutations.…”