CRISPR/Cas9-mediated targeted mutagenesis in grape.

Ikuko Nakajima,Noriyuki Onoue,Takaya Moriguchi,Toshiya Yamamoto,Akifumi Azuma,Yusuke Ban,Masaki Endo,Seiichi Toki,Sara Amancio

doi:10.1371/journal.pone.0177966

Abstract

RNA-guided genome editing using the CRISPR/Cas9 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has been applied successfully in several plant species. However, to date, there are few reports on the use of any of the current genome editing approaches in grape—an important fruit crop with a large market not only for table grapes but also for wine. Here, we report successful targeted mutagenesis in grape (Vitis vinifera L., cv. Neo Muscat) using the CRISPR/Cas9 system. When a Cas9 expression construct was transformed to embryonic calli along with a synthetic sgRNA expression construct targeting the Vitis vinifera phytoene desaturase (VvPDS) gene, regenerated plants with albino leaves were obtained. DNA sequencing confirmed that the VvPDS gene was mutated at the target site in regenerated grape plants. Interestingly, the ratio of mutated cells was higher in lower, older, leaves compared to that in newly appearing upper leaves. This result might suggest either that the proportion of targeted mutagenized cells is higher in older leaves due to the repeated induction of DNA double strand breaks (DSBs), or that the efficiency of precise DSBs repair in cells of old grape leaves is decreased.

Highlights

Functional analysis of any gene of interest in plant genomes has traditionally relied heavily on the use of transfer DNA (T-DNA) and transposon insertional mutagenesis, or chemical- and irradiation-induced mutagenesis, to generate mutants
To test whether CRISPR/Cas9-mediated targeted mutagenesis system functions in grape, four different sites on Vitis vinifera phytoene desaturase (VvPDS) exons were targeted for cleavage (Fig 2a and 2b)
These target sequences were selected by first identifying the NGG protospacer adjacent motif (PAM) sequence required for Streptococcus pyogenes Cas9, and capturing the 20 nucleotides immediately upstream of the PAM sequence for use as the spacer in the single guide RNAs (sgRNAs)

Summary

Introduction

Functional analysis of any gene of interest in plant genomes has traditionally relied heavily on the use of transfer DNA (T-DNA) and transposon insertional mutagenesis, or chemical- and irradiation-induced mutagenesis, to generate mutants. Saturation mutagenesis is difficult in non-model plants due to a lack of genome information, large genome size and/or low transformation efficiency. Recent progress in the use of sequence-specific nucleases (SSNs) has opened new opportunities for reverse genetics in plants. SSNs, which include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered.

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Conclusion