Highly efficient genome editing in Xanthomonas oryzae pv.oryzae through repurposing the endogenous type I-CCRISPR-Cas system.

Abstract

Efficient and modular genome editing technologies that manipulate the genome of bacterial pathogens will facilitate the study of pathogenesis mechanisms. However, such methods are yet to be established for Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of rice bacterial blight. We identified a single type I‐C CRISPR‐Cas system in the Xoo genome and leveraged this endogenous defence system for high‐efficiency genome editing in Xoo. Specifically, we developed plasmid components carrying a mini‐CRISPR array, donor DNA, and a phage‐derived recombination system to enable the efficient and programmable genome editing of precise deletions, insertions, base substitutions, and gene replacements. Furthermore, the type I‐C CRISPR‐Cas system of Xoo cleaves target DNA unidirectionally, and this can be harnessed to generate large genomic deletions up to 212 kb efficiently. Therefore, the genome‐editing strategy we have developed can serve as an excellent tool for functional genomics of Xoo, and should also be applicable to other CRISPR‐harbouring bacterial plant pathogens.