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

摘要: CRISPR/Cas9-mediated base editors, based on cytidine deaminase or adenosine deaminase, are emerging genetic technologies that facilitate genomic manipulation in many organisms. Since 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 Streptomyces, method been successfully applied several model and non-model species, Streptomyces coelicolor griseofuscus. this study, we first proved BE2 (rAPOBEC1-dCas9-UGI) BE3 (rAPOBEC1-nCas9-UGI) were functional tools lividans 66, albeit with much efficiency S. coelicolor. Uracil generated deamination key intermediate process, can be hydrolyzed by uracil glycosidase (UDG) involved intracellular excision repair, resulting low efficiency. By knocking out two endogenous UDGs (UDG1 UDG2), managed improve 3.4-67.4-fold among different loci. However, inactivation UDG detrimental stability future application engineered strains. Therefore, finally developed nti s ense RNA interference-enhanced CRISPR/Cas9 B ase E diting (asRNA-BE) transiently disrupt expression glycosidases during editing, leading 2.8-65.8-fold enhanced better stability. Our results demonstrate asRNA-BE tool for 66 might beneficial improving other