Beijing Key Laboratory of Grape Science and Enology and Key Laboratory of Plant Resource, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, PR China.
University of Chinese Academy of Sciences, Beijing 100049, PR China.
Sci Rep. 2016 Aug 31;6:32289. doi: 10.1038/srep32289.
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.
CRISPR/Cas9 系统（CRISPR 相关蛋白 9）已成功应用于多个植物物种的靶基因编辑。然而，该系统是否可用于葡萄的基因组编辑尚不清楚。本研究通过 CRISPR/Cas9 系统描述了‘霞多丽’悬浮细胞和植株的基因组编辑和靶向基因突变。设计了两个单向导 RNA（sgRNA）以靶向 L-山梨糖脱氢酶基因（IdnDH）的不同位点。CEL I 内切酶分析和测序结果显示靶位点出现预期的插入缺失突变，在转基因细胞群（CM）以及表达 sgRNA1/Cas9 的相应再生植株中观察到 100%的突变频率。在转基因 CM 中检测到的大多数突变是 1 个碱基的插入，其次是 1 到 3 个核苷酸的缺失。通过测序潜在的脱靶位点也评估了脱靶活性，未检测到明显的脱靶事件。我们的研究结果表明，CRISPR/Cas9 系统是一种高效、特异的葡萄精确基因组编辑工具。
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