Development of a DNA double-strand break-free base editing tool in Corynebacterium glutamicum for genome editing and metabolic engineering

摘要

As a traditional amino acid producing bacterium, Corynebacterium glutamicum is a platform strain for production of various fine chemicals. Based on the CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas9 system, gene editing tools that enable base conversion in the genome of C.?glutamicum have been developed. However, some problems such as genomic instability caused by DNA double-strand break (DSB) and off-target effects need to be solved. In this study, a DSB-free single nucleotide genome editing system was developed by construction of a bi-directional base conversion tool TadA-dCas9-AID. This system includes cytosine base editors (CBEs): activation-induced cytidine deaminase (AID) and adenine deaminase (ABEs): tRNA adenosine deaminase (TadA), which can specifically target the gene through a 20-nt single guide RNA (sgRNA) and achieve the base conversion of C-T, C-G and A-G in the 28-bp editing window upstream of protospacer adjacent motif. Finally, as a proof-of-concept demonstration, the system was used to construct a mutant library of zwf gene in C.?glutamicum S9114 genome to improve the production of a typical nutraceutical N -acetylglucosamine (GlcNAc). The GlcNAc titer of the mutant strain K293R was increased by 31.9% to 9.1??g/L in shake flask. Here, the developed bases conversion tool TadA-dCas9-AID does not need DNA double-strand break and homologous template, and is effective for genome editing and metabolic engineering in C.?glutamicum .