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Article|01 May 2021|OPEN
Whole-genome sequencing reveals rare off-target mutations in CRISPR/Cas9-edited grapevine
Xianhang Wang1,2, Mingxing Tu1,3, Ya Wang1,3, Wuchen Yin1,3, Yu Zhang4, Hongsong Wu4, Yincong Gu5, Zhi Li1,3, Zhumei Xi2 & Xiping Wang1,3,
1State Key Laboratory of Crop Stress Biology in Arid Areas, College of Horticulture, Northwest A&F University, 712100 Yangling, Shaanxi, China
2College of Enology, Northwest A&F University, 712100 Yangling, Shaanxi, China
3Key Laboratory of Horticultural Plant Biology and Germplasm Innovation in Northwest China, Ministry of Agriculture, Northwest A&F University, 712100 Yangling, Shaanxi, China
4Novogene Technologies Corporation, 100000 Beijing, China
5OEbiotech Corporation, 200000 Shanghai, China

Horticulture Research 8,
Article number: 114 (2021)
doi: 10.1038/hortres.2021.114
Views: 194

Received: 21 Nov 2020
Revised: 03 Mar 2021
Accepted: 14 Mar 2021
Published online: 01 May 2021


The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) system is a powerful tool for targeted genome editing, with applications that include plant biotechnology and functional genomics research. However, the specificity of Cas9 targeting is poorly investigated in many plant species, including fruit trees. To assess the off-target mutation rate in grapevine (Vitis vinifera), we performed whole-genome sequencing (WGS) of seven Cas9-edited grapevine plants in which one of two genes was targeted by CRISPR/Cas9 and three wild-type (WT) plants. In total, we identified between 202,008 and 272,397 single nucleotide polymorphisms (SNPs) and between 26,391 and 55,414 insertions/deletions (indels) in the seven Cas9-edited grapevine plants compared with the three WT plants. Subsequently, 3272 potential off-target sites were selected for further analysis. Only one off-target indel mutation was identified from the WGS data and validated by Sanger sequencing. In addition, we found 243 newly generated off-target sites caused by genetic variants between the Thompson Seedless cultivar and the grape reference genome (PN40024) but no true off-target mutations. In conclusion, we observed high specificity of CRISPR/Cas9 for genome editing of grapevine.