The application of the CRISPR-Cas9 system in pv. actinidiae.

pv. actinidiae (Psa) has emerged as a major bacterial pathogen of kiwifruit cultivation throughout the world. We aim to introduce a CRISPR-Cas9 system, a commonly used genome editing tool, into Psa. The protocols may also be useful in other species. Using standard molecular biology techniques, we modified plasmid pCas9, which carries the CRISPR-Cas9 sequences from for use in Psa. The final plasmid, pJH1, was produced in a series of steps and is maintained with selection in both and Psa. We have constructed plasmids carrying a CRISPR-Cas9 system based on that of , which can be maintained, under selection, in Psa. We have shown that the gene targeting capacity of the CRISPR-Cas9 system is active and that the Cas9 protein is able to cleave the targeted sites. The Cas9 was directed to several different sites in the genome. Using Cas9 we have generated Psa transformants that no longer carry the native plasmid present in Psa, and other transformants that lack the integrative, conjugative element, Pac_ICE1. Targeting of a specific gene, a chromosomal non-ribosomal peptide synthetase, led to gene knockouts with the transformants having deletions encompassing the target site. We have constructed shuttle plasmids carrying a CRISPR-Cas9 system that are maintained in both and pv. actinidiae. We have used this gene editing system to eliminate features of the accessory genome (plasmids or ICEs) from Psa and to target a single chromosomal gene.