CRISPR/Cas9-mediated efficient targeted mutagenesis in Chardonnay (Vitis vinifera L.)

Abstract

The type II clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system (CRISPR/Cas9) has been successfully applied to edit target genes in multiple plant species. However, it remains unknown whether this system can be used for genome editing in grape. In this study, we described genome editing and targeted gene mutation in ‘Chardonnay’ suspension cells and plants via the CRISPR/Cas9 system. Two single guide RNAs (sgRNAs) were designed to target distinct sites of the L-idonate dehydrogenase gene (IdnDH). CEL I endonuclease assay and sequencing results revealed the expected indel mutations at the target site, and a mutation frequency of 100% was observed in the transgenic cell mass (CM) as well as corresponding regenerated plants with expression of sgRNA1/Cas9. The majority of the detected mutations in transgenic CM were 1-bp insertions, followed by 1- to 3-nucleotide deletions. Off-target activities were also evaluated by sequencing the potential off-target sites, and no obvious off-target events were detected. Our results demonstrated that the CRISPR/Cas9 system is an efficient and specific tool for precise genome editing in grape.

Cultivar-specific gene modulation in Vitis vinifera: analysis of the promoters regulating the expression of WOX transcription factors

“…Indeed, the characterisation of VvWOX promoters has provided new information about tissue- and time-specific promoters useful for functional studies and as an alternative to traditional constitutive promoters in cis-genetic approaches. In addition, these results also provide interesting information for genome editing in grapevine749, suggesting that point mutations in regulatory sequences could be determinant for increasing or decreasing gene transcription.…”

“…In the last years, there has been a new impetus to study promoters in grapevine45, which arises from the applications of new “Sustainable Biotechnology”, i.e. cis-genesis and genome editing67, for which it is very important to know and access many regulatory sequences.…”

DNA-Free Genetically Edited Grapevine and Apple Protoplast Using CRISPR/Cas9 Ribonucleoproteins

“…CRISPR/Cas9 editing tools are efficiently exploited for gene mutation, repression, activation and epigenome editing in several model and crop plants, such as Arabidopsis, tobacco, rice, sorghum, maize, wheat, poplar, tomato, soya bean, petunia, citrus and recently grape and apple (Nishitani et al, 2016; Ren et al, 2016; Song et al, 2016). Meanwhile CRISPR/Cas9 RNPs DNA-free genome editing tools are successfully demonstrated in Arabidopsis , tobacco, lettuce, rice, petunia, and recently in wheat (Woo et al, 2015; Subburaj et al, 2016; Zhang et al, 2016).…”

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

“…The CRISPR-Cas9 system has been successfully applied to an increasing range of model and crop species [7,18,19]. However, it is still limited to plants for which efficient transformation and regeneration protocols are available.…”

“…However, some of these techniques are still in their early developments in plants, and gene knock-in, in particular, needs to be optimized for many species. Indeed, whereas the knock-out of endogenous plant loci is generally easy to achieve (with mutagenesis efficiencies varying from 2% [6] to 100% [7] of mutated individuals), CRISPR-induced gene knock-in of a donor DNA by homology-driven repair (HDR) is much more difficult to perform in higher plants, with efficiencies rarely reaching a few percent [2,6,[8][9][10][11]. It explains why among the CRISPR-induced traits already reported in the literature, such as diseases tolerances [12][13][14], adaptation to drought [2], modified ripening profiles [15], male sterility and factors impacting yield [9,16,17], most of them are derived from gene knock-out, and still very few from gene knock-in.…”