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

• Published in: Plant biotechnology journal Vol. 17; no. 5; pp. 858 - 868
• Main Authors: 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
• Format: Journal Article
• Language: English
• Published: England John Wiley & Sons, Inc 01.05.2019; John Wiley and Sons Inc
• Subjects: biotechnology; Cell division; Comparative analysis; Cotton; CRISPR; CRISPR-Associated Protein 9; CRISPR-Cas Systems; CRISPR/Cas9; Crop improvement; DNA sequencing; Gene Editing; Gene sequencing; Genes; Genetic aspects; Genetic diversity; Genetic Variation - genetics; Genome, Plant - genetics; Genome-Wide Association Study; Genomes; Genomics; Gossypium - genetics; Mutation; Mutation - genetics; Nucleotide sequencing; Nucleotides; off‐target; Plants; Plants, Genetically Modified - genetics; pre‐existing/inherent variation; Proteins; R&D; Research & development; Single nucleotide polymorphisms; Single-nucleotide polymorphism; Software; Somaclonal variation; Target recognition; Transcription factors; Whole Genome Sequencing; whole genome sequencing; cotton; pre-existing/inherent variation; somaclonal variation; CRISPR/Cas9; off-target
• ISSN: 1467-7644; 1467-7652; 1467-7652
• DOI: 10.1111/pbi.13020

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 (SNPs) 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 sgRNA 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 PAMs, which suggested great care should be taken to design sgRNA for the minimizing of off‐target effect. These findings suggested that CRISPR/Cas9 system is highly specific for cotton plants.