Precise A∙T to G∙C base editing in the allotetraploid rapeseed (Brassica napus L.) genome.

Rapeseed is an important source of oilseed crop in the world. Achieving genetic improvement has always been the major goal in rapeseed production. Single nucleotide substitution is the basis of most genetic variation underpinning important agronomic traits. Nowadays, Cas-base editing acts as an efficient tool to mediate single-base substitution at the target site. In this study, four adenine base editors (ABE) were modified to achieve adenosine base editing at different genome sites in allotetraploid Brassica napus. We designed 18 small guide RNAs to target phytoene desaturase (PDS), acetolactate synthase (ALS), CLAVATA3 (CLV3), CLV2, TRANSPARENT TESTA12 (TT12), carotenoid isomerase (CRTISO), designated de-etiolated-2 (DET2), BRANCHED1 (BRC1), zeaxanthin epoxidase (ZEP) genes, respectively. Among the four ABE systems, pBGE17 had the highest base-editing efficiency, with an average editing efficiency of 3.51%. Target sequencing results revealed that the editing window ranged from A5 to A8 of the protospacer-adjacent motif (PAM) sequence. Moreover, the ABEmax-nCas9NG system with NG PAM was developed, with a base-editing efficiency of 1.22%. These results revealed that ABE system developed in this study could efficiently induce A to G substitution and the ABE-nCas9NG system could broaden editing window in oilseed rape.