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

Li, J.; Manghwar, H.; Sun, L.; Wang, P.; Wang, G.; Sheng, H.; Zhang, J.; Liu, H.; Qin, L.; Rui, H.; Lindsey, K.; Daniell, H.; Jin, S.; Zhang, X.

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


Authors

J. Li

H. Manghwar

L. Sun

P. Wang

G. Wang

H. Sheng

J. Zhang

H. Liu

L. Qin

H. Rui

H. Daniell

S. Jin

X. Zhang



Abstract

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 3 negative (Ne) control and 3 wild‐type (WT) plants. In total, 4,188 ‐ 6,404 unique single nucleotide polymorphisms (SNPs) and 312 ‐ 745 insertions/deletions (indels) were detected in 14 Cas9‐edited plants compared with 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/natural variation from maternal plants, but not off‐target effects. Of a total of 4,413 potential off‐target sites (allowing ≤ 5 mismatches within the 20‐bp sgRNA and 3‐bp PAM sequences), the WGS data revealed that only 4 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.

Journal Article Type Article
Acceptance Date Oct 1, 2018
Online Publication Date Oct 30, 2018
Publication Date May 31, 2019
Deposit Date Oct 3, 2018
Publicly Available Date Nov 6, 2018
Journal Plant Biotechnology Journal
Print ISSN 1467-7644
Electronic ISSN 1467-7652
Publisher Association of Applied Biologists
Peer Reviewed Peer Reviewed
Volume 17
Issue 5
Pages 858-868
DOI https://doi.org/10.1111/pbi.13020
Public URL https://durham-repository.worktribe.com/output/1317703

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Publisher Licence URL
http://creativecommons.org/licenses/by/4.0/

Copyright Statement
Advance online version © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.






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