Abstract
AbstractCRISPR-based genome editing technology is revolutionizing prokaryotic research, but it has been rarely studied in bacterial plant pathogens. Here, we have developed a targeted genome editing method with no requirement of donor templates for convenient and efficient gene knockout inXanthomonas oryzaepv.oryzae(Xoo), one of the most important bacterial pathogens on rice, by employing the heterogenous CRISPR/Cas12a fromFrancisella novicidaand NHEJ proteins fromMycobacterium tuberculosis.FnCas12a nuclease generated both small and large DNA deletions at the target sites as well as it enabled multiplex genome editing, gene cluster deletion and plasmid cure in theXooPXO99Astrain. Accordingly, a non-TAL effector-free polymutant strain PXO99AD25E, which lacks all 25Xopgenes involved inXoopathogenesis, has been engineered through iterative genome editing. Whole-genome sequencing analysis indicated that FnCas12a did not have a noticeable off-target effect. In addition, we revealed that these strategies are also suitable for targeted genome editing in another bacterial plant pathogenPseudomonas syringaepv.tomato(Pst). We believe that our bacterial genome editing method will greatly expand the CRISPR study on microorganisms and advance our understanding of the physiology and pathogenesis ofXoo.
Publisher
Cold Spring Harbor Laboratory