Development And Application Of Base Editing Technology For Actinomycetales

Base editing technology based on CRISPR/Cas system has been widely used by researchers in mammalian cells,plants and microorganisms.In order to expand the application of the base editing system in actinomycetes,PmCDA1 from lampreys was fused with d/nCas9 and UGI to construct a series of Target-AID base editors,but the editors were not functional in Streptomyces lividans.After that,rAPOBEC1 was fused with d/nCas9 protein and UGI protein,contributing to successful conversion of C to T in Streptomyces coelicolor,Streptomyces lividans and Corynebacterium glutamate of actinomycetales.Both the BE2-type cytosine base editors(rAPOBEC1-XTEN-dCas9(D10A)-UGI)and the BE3-type base editors(rAPOBEC1-XTEN-nCas9(D10A)-UGI)could achieve efficient editing in Streptomyces coelicolor,but the editing efficiency was very low in the parental Streptomyces lividans.By knocking out uracil DNA glycosylase inhibitor gene udgl on the genome of Streptomyces lividans,the editing efficiency of the third and fifth sites of the target redN gene increased from 3%to 42.75%and 10.9%to 65.75%,respectively.The rAPOBEC1-XTEN-nCas9(D10A)editor couldalso achieve C to T base substitution in Corynebacterium glutamicum,but the editing efficiency was very low,and C to A or C to G editing events occurs,with poor editing specificity.BE3 cytosine editor with UGI protein fused to the N-terminal of rAPOBEC1-XTEN-nCas9 protein,was constructed,and its editing efficiency in Corybacterium glutamicum was up to 100%.There was only C to T base substitution with high editing specificity.For convenience in future applications,the dual-plasmid base editing system was simplified to single-plasmid system,and the transformation efficiency was enhanced.Finally,the single-plasmid based BE3 base editorwas tested at other genomic loci,and exhibited high editing efficiency and broad editing window(-11 to-19 positions upstream of the PAM sequence).which would be beneficial to cover more target genomic loci.This work would enrich genome editing toolkit for actinomycetales engineering,and provide reference for genome editing in other actinomycetales species.