Abstract:

Base editing is a new genome editing technology, and has the advantages of not producing double strand break, not relying on homologous recombination and not adding foreign template. It has been widely developed and applied in eukaryotes and prokaryotes. In order to further expand the genome coverage of base editing technology in Corynebacterium glutamicum, three novel Cas9 mutants or different Cas9 proteins with relaxed PAM restriction were applied to cytosine base editing tools, namely, SpRY mutant（NRN> NYN PAM）, SpG mutant（NGN PAM）and ScCas9⁺⁺ protein（NNG PAM）, the PAM extension for base gene editing tool is achieved. The base editing system combined with SpRY mutant showed more relaxed PAM recognition. Except for CAT, CAC and TAA PAMs, other NRN PAMs were recognized to varying degrees, but the overall editing efficiency was low, which was difficult for widely application. The base editing system combined with SpG mutant edited genome loci with all NGN PAMs, and the editing efficiency was better than that of SpRY mutant, but the editing efficiency of NGG PAM sites reduced by 9.3%-55.9% compared with that of original Cas9 protein. In combination with the base editing system of ScCas9⁺⁺ protein, except for the genome loci of TCG and CTG PAM, the genome loci of other tested NNG PAM can be edited, and the genome editing efficiency of most loci was high, and the highest editing efficiency reached 100%. This study not only helps base editing tools to cover more genomic sites in Corynebacterium glutamicum, but also provides a favorable reference for PAM expansion of other genome editing tools based on CRISPR/Cas system.

Key words: cytosine base editing, Corynebacterium glutamicum, PAM extension, CRISPR/Cas