Whole genome sequencing reveals rare off‐target mutations and considerable inherent genetic or/and somaclonal variations in CRISPR/Cas9‐edited cotton plants

• DOI: 10.1111/pbi.13020
• Publication Date: 2018-10-06T14:38:04Z
• Authors: Jianying Li; Hakim Manghwar; Lin Sun; Pengcheng Wang; Guanying Wang; Hanyan Sheng; Jie Zhang; Hao Liu; Lei Qin; Hangping Rui; Bo Li; Keith Lindsey; Henry Daniell; Shuangxia Jin; Xianlong Zhang
• Keywords: Indel; Sanger sequencing; Somaclonal variation; Subgenomic mRNA

Summary The CRISPR /Cas9 system has been extensively applied for crop improvement. However, our understanding of Cas9 specificity is very limited in Cas9‐edited plants. To identify on‐ and off‐target mutation an edited crop, we described whole genome sequencing ( WGS ) 14 cotton plants targeted to three genes, negative (Ne) control wild‐type WT In total, 4188–6404 unique single‐nucleotide polymorphisms SNP s) 312–745 insertions/deletions (indels) were detected compared , reference sequences. Since the majority these variations lack a protospacer‐adjacent motif PAM ), demonstrated that most following are due either somaclonal variation or/and pre‐existing/inherent from maternal plants, but not effects. Of total 4413 potential sites (allowing ≤5 mismatches within 20‐bp sg RNA 3‐bp sequences), data revealed only four bona fide indel mutations, validated by Sanger sequencing. Moreover, inherent genetic can generate novel destroy s, which suggested great care should be taken design minimizing effect. These findings highly specific