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

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.

Conventional and Molecular Techniques from Simple Breeding to Speed Breeding in Crop Plants: Recent Advances and Future Outlook

“…The base-editing approach using CRISPR/nCas9 (Cas9 nickase) or dCas9 (deactivated Cas9) fused with cytidine deaminase is a powerful tool to create point mutations. In this study, we point out the remarkable G. hirsutum-base editor 3 (GhBE3) base enhancing system developed to create single base mutations in the allotetraploid genome of cotton (Gossypium hirsutum) [101].…”

Gossypium Genomics: Trends, Scope, and Utilization for Cotton Improvement

“…With the development of modern technologies, plant breeders have the opportunity to overcome this bottleneck. Studies have demonstrated that targeted mutagenesis and precise base editing can be accurately applied for the G. hirsutum genome using genome editing systems [68][69][70][71][72][73][74][75][76][77]. Genome resequencing has identified many domestication genes associated with fiber quality and yield traits.…”

Simplified adenine base editors improve adenine base editing efficiency in rice

“…Cytosine base editor (CBE), the first member in the base editing toolbox, can convert cytidine (C) to thymidine (T) (or G to A) at specific genomic sites in an sgRNA‐dependent manner (Komor et al ., ). The most commonly used CBEs in plants consist of a Cas9 nickase (nSpCas9 (D10A)) fused with a cytidine deaminase and a uracil glycosylase inhibitor (UGI) (Li et al ., ; Lu and Zhu, ; Qin et al ., ; Shimatani et al ., ; Zong et al ., ). Although the CBEs have been shown to be functional in many plant species, the narrow target range and low editing efficiency considerably limit their application in crop breeding.…”