Antisense RNA Interference-Enhanced CRISPR/Cas9 Base Editing Method for Improving Base Editing Efficiency in 66.

CRISPR/Cas9-mediated base editors, based on cytidine deaminase or adenosine deaminase, are emerging genetic technologies that facilitate genomic manipulation in many organisms. Since base editing is free from DNA double-strand breaks (DSBs), it has certain advantages, such as a lower toxicity, compared to the traditional DSB-based genome engineering technologies. In terms of , a base editing method has been successfully applied in several model and non-model species, such as and . In this study, we first proved that BE2 (rAPOBEC1-dCas9-UGI) and BE3 (rAPOBEC1-nCas9-UGI) were functional base editing tools in 66, albeit with a much lower editing efficiency compared to that of . Uracil generated in deamination is a key intermediate in the base editing process, and it can be hydrolyzed by uracil DNA glycosidase (UDG) involved in the intracellular base excision repair, resulting in a low base editing efficiency. By knocking out two endogenous UDGs (UDG1 and UDG2), we managed to improve the base editing efficiency by 3.4-67.4-fold among different loci. However, the inactivation of UDG is detrimental to the genome stability and future application of engineered strains. Therefore, we finally developed ntiense interference-enhanced CRISPR/Cas9 ase diting method (asRNA-BE) to transiently disrupt the expression of uracil DNA glycosidases during base editing, leading to a 2.8-65.8-fold enhanced editing efficiency and better genome stability. Our results demonstrate that asRNA-BE is a much better editing tool for base editing in 66 and might be beneficial for improving the base editing efficiency and genome stability in other strains.