Abstract: The base editing technology,combining the targeted specificity of CRISPR/Cas system and the catalytic activity of cytidine deaminase,has been developed and widely applied in mammalian cells,plants and microorganism,due to not introducing double-stranded DNA break,a DNA template and relying on host homologous recombination repair pathway. In order to expand the application of base editing in Corynebacterium glutamicum,C to T conversion was achieved by fusing the cytidine deaminase（rAPOBEC1）and nCas9;however,the initial editing efficiency was low（0 to 20%）. The base editor BE3 was then constructed by adding the UGI protein in the C terminus of the rAPOBEC1-nCas9 fusion,which inhibited the DNA base excise repair pathway and significantly improved the editing performance with the C to T conversion efficiency up to 90%. For convenience in future applications,the dual-plasmid base editing system was simplified to single-plasmid system,and the transformation efficiency was remarkably enhanced. Finally,by using other genomic loci as editing target,the single-plasmid based BE3 base editor was proved to be powerful with high editing efficiency and broad editing window（-11 to -19 positions upstream of the PAM sequence）,which was beneficial to cover more target genomic loci and provided more tools for genome engineering of C. glutamicum.

Key words: base editing, Corynebacterium glutamicum, CRISPR/Cas9, cytidine deaminase