Base editing with high efficiency in allotetraploid oilseed rape by A3A-PBE system

CRISPR/Cas-base editing is an emerging technology that could convert a nucleotide to another type at the target site. In this study, A3A-PBE system consisting of human A3A cytidine deaminase fused with a Cas9 nickase and uracil glycosylase inhibitor was established and developed in allotetraploidBrassica napus. We designed three sgRNAs to targetALS,RGAandIAA7genes, respectively. Base-editing efficiency was demonstrated to be more than 20% for all the three target genes. Target sequencing results revealed that the editing window ranged from C1 to C10 of the PAM sequence. Base-edited plants ofALSconferred high herbicide resistance, while base-edited plants ofRGAorIAA7exhibited decreased plant height. All the base editing could be genetically inherited from T(0)to T(1)generation. Several Indel mutations were confirmed at the target sites for all the three sgRNAs. Furthermore, though no C to T substitution was detected at the most potential off-target sites, large-scale SNP variations were determined through whole-genome sequencing between some base-edited and wild-type plants. These results revealed that A3A-PBE base-editing system could effectively convert C to T substitution with high-editing efficiency and broadened editing window in oilseed rape. Mutants forALS,IAA7andRGAgenes could be potentially applied to confer herbicide resistance for weed control or with better plant architecture suitable for mechanic harvesting.

Automated summary

The study established an A3A-PBE base-editing system in allotetraploid Brassica napus, demonstrating high editing efficiency and broadened editing window. Mutants for ALS, IAA7, and RGA genes showed different traits, with potential applications in herbicide resistance or plant architecture improvement. Target sequencing identified an editing window from C1 to C10 and confirmed base-editing inheritance from T(0) to T(1) generations with several Indel mutations at target sites.