Construction of Erwinia amylovora mutants via crRNA expression

Abstract Genome editing is essential for studying gene function for pathogens such as Erwinia amylovora , which causes devastating fire blight in fruit trees of the family Rosaceae. CRISPR/Cas genome editing technology has been widely used in various model organisms, but E. amylovora lacks effective genome editing tools. Here, we present a simple and effective gene knockout method based on the E. amylovora endogenous type I-E CRISPR/Cas system that uses plasmid to express crRNA. The plasmid contains the CRR1 array expression cassette of E. amylovora , the replication initiation region of the E. amylovora plasmid pEA60, and backbone of the cloning vector pMD19. Using this method, we successfully obtained the pEA29 plasmid curing and ams operon deletion mutants. Five distinct deletions (~ 33 k to ~ 52 k bp) were identified from 11 plasmid-transformed clones by whole-genome sequencing, with no off-target effects detected. The ams operon knockout mutant, as expected, did not produce amylovoran, the major extracellular polysaccharide component and pathogenic factor of E. amylovora , and was non-pathogenic in plants. Thus, this method allows for quick and convenient construction of genetic mutants of E. amylovora using plasmid-expressed crRNAs.