Open AccessWhole genome sequencing reveals rare off‐target mutations and considerable inherent genetic or/and somaclonal variations in CRISPR /Cas9‐edited cotton plants
Author(s) -
Li Jianying,
Manghwar Hakim,
Sun Lin,
Wang Pengcheng,
Wang Guanying,
Sheng Hanyan,
Zhang Jie,
Liu Hao,
Qin Lei,
Rui Hangping,
Li Bo,
Lindsey Keith,
Daniell Henry,
Jin Shuangxia,
Zhang Xianlong
Publication year - 2019
Publication title -
plant biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.525
H-Index - 115
eISSN - 1467-7652
pISSN - 1467-7644
DOI - 10.1111/pbi.13020
Subject(s) - indel , biology , crispr , sanger sequencing , cas9 , genetics , somaclonal variation , genome , computational biology , subgenomic mrna , deep sequencing , single nucleotide polymorphism , whole genome sequencing , gene , dna sequencing , genotype , tissue culture , in vitro
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 in an edited crop, we described whole genome sequencing ( WGS ) of 14 Cas9‐edited cotton plants targeted to three genes, and three negative (Ne) control and three wild‐type ( WT ) plants. In total, 4188–6404 unique single‐nucleotide polymorphisms ( SNP s) and 312–745 insertions/deletions (indels) were detected in 14 Cas9‐edited plants compared to WT , negative and cotton reference genome sequences. Since the majority of these variations lack a protospacer‐adjacent motif ( PAM ), we demonstrated that the most variations following Cas9‐edited are due either to somaclonal variation or/and pre‐existing/inherent variation from maternal plants, but not off‐target effects. Of a total of 4413 potential off‐target sites (allowing ≤5 mismatches within the 20‐bp sg RNA and 3‐bp PAM sequences), the WGS data revealed that only four are bona fide off‐target indel mutations, validated by Sanger sequencing. Moreover, inherent genetic variation of WT can generate novel off‐target sites and destroy PAM s, which suggested great care should be taken to design sg RNA for the minimizing of off‐target effect. These findings suggested that CRISPR /Cas9 system is highly specific for cotton plants.