High-efficient and precise base editing of C•G to T•A in the allotetraploid cotton (Gossypium hirsutum) genome using a modified CRISPR/Cas9 system.

Lei Qin,Tianliang Lu,Xianlong Zhang,Lin Sun,Hangping Rui,Keith Lindsey,Bo Li,Qiongqiong Wang,Shuangxia Jin,Huimei Huang,Jianying Li,Xiao Ding,Zhongping Xu,Henry Daniell,Muna Alariqi,Hakim Manghwar,Guanyin Wang

doi:10.1111/pbi.13168

Abstract

SummaryThe base‐editing technique using CRISPR/nCas9 (Cas9 nickase) or dCas9 (deactivated Cas9) fused with cytidine deaminase is a powerful tool to create point mutations. In this study, a novel G. hirsutum‐Base Editor 3 (GhBE3) base‐editing system has been developed to create single‐base mutations in the allotetraploid genome of cotton (Gossypium hirsutum). A cytidine deaminase sequence (APOBEC) fused with nCas9 and uracil glycosylase inhibitor (UGI) was inserted into our CRISPR/Cas9 plasmid (pRGEB32‐GhU6.7). Three target sites were chosen for two target genes, GhCLA and GhPEBP, to test the efficiency and accuracy of GhBE3. The editing efficiency ranged from 26.67 to 57.78% at the three target sites. Targeted deep sequencing revealed that the C→T substitution efficiency within an ‘editing window’, approximately six‐nucleotide windows of −17 to −12 bp from the PAM sequence, was up to 18.63% of the total sequences. The 27 most likely off‐target sites predicted by CRISPR‐P and Cas‐OFFinder tools were analysed by targeted deep sequencing, and it was found that rare C→T substitutions (average < 0.1%) were detected in the editing windows of these sites. Furthermore, whole‐genome sequencing analyses on two GhCLA‐edited and one wild‐type plants with about 100× depth showed that no bona fide off‐target mutations were detectable from 1500 predicted potential off‐target sites across the genome. In addition, the edited bases were inherited to T1 progeny. These results demonstrate that GhBE3 has high specificity and accuracy for the generation of targeted point mutations in allotetraploid cotton.

Highlights

Single-nucleotide mutation is the basis of much genetic variation underpinning important agronomic traits in crop plants
We developed an efficient and precise base editor system (GhBE3) consisting of a cytidine deaminase domain fused with nCas9 and uracil glycosylase inhibitor (UGI), for use in allotetraploid cotton, and which exhibits a high base-editing efficiency with no detectable offtargets effects
According to specific barcode primers designed for a single strain, high-throughput data of target point mutation detection were sorted into each single strain

Summary

The base-editing technique using CRISPR/nCas (Cas nickase) or dCas (deactivated Cas9) fused with cytidine deaminase is a powerful tool to create point mutations. The 27 most likely off-target sites predicted by CRISPR-P and Cas-OFFinder tools were analysed by targeted deep sequencing, and it was found that rare C?T substitutions (average < 0.1%) were detected in the editing windows of these sites. Whole-genome sequencing analyses on two GhCLA-edited and one wild-type plants with about 1009 depth showed that no bona fide offtarget mutations were detectable from 1500 predicted potential off-target sites across the genome. The edited bases were inherited to T1 progeny These results demonstrate that GhBE3 has high specificity and accuracy for the generation of targeted point mutations in allotetraploid cotton

Introduction

Experimental procedures

Results and discussion