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

Chong Ren,Zhenchang Liang,Zhan Zhang,Shaohua Li,Xianju Liu,Yi Wang,Wei Duan

doi:10.1038/srep32289

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.

Highlights

Targeted genome editing (TGE) using site-specific nucleases (SSNs) promises to be critical to basic and applied biology research
The Arabidopsis U6 promoter (AtU6) and single guide RNAs (sgRNAs) were first combined by PCR, and the sgRNA expression cassette with adaptors was inserted into the linearized vector by the homologous recombination (HR) method
Two binary vectors were constructed, where the expression of Cas[9] and sgRNA was driven by the CaMV 35S promoter and AtU6, respectively (Fig. 1C)

Summary

Introduction

Targeted genome editing (TGE) using site-specific nucleases (SSNs) promises to be critical to basic and applied biology research These powerful SSN tools, comprising zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system (CRISPR/Cas9), introduce targeted DNA double-strand breaks (DSBs) and subsequently trigger DNA repair pathways involving either non-homologous end-joining (NHEJ) or homologous recombination (HR)[1]. Somatic mutations frequently occur during the tissue culture-based transformation and many abnormal plantlets may be generated from somatic embryos[38,39] It is difficult and time-consuming to obtain enough grapevines for our studies, so ‘Chardonnay’ suspension cells were adopted as our main experimental material, and the L-idonate dehydrogenase gene (IdnDH, LOC100232980), which controls the biosynthesis of tartaric acid (TA) in grape[40], was chosen as the target gene to be disrupted site- in suspension cells of ‘Chardonnay’. Our data demonstrated that the CRISPR/Cas[9] system could efficiently induce targeted mutations in ‘Chardonnay’ suspension cells as well as its regenerated plants, indicating the feasibility of this system as a powerful tool for genome modification in grape

Methods

Results

Conclusion