Genome editing of Ralstonia eutropha using an electroporation-based CRISPR-Cas9 technique

is an important bacterium for the study of polyhydroxyalkanoates (PHAs) synthesis and CO fixation, which makes it a potential strain for industrial PHA production and attractive host for CO conversion. Although the bacterium is not recalcitrant to genetic manipulation, current methods for genome editing based on group II introns or single crossover integration of a suicide plasmid are inefficient and time-consuming, which limits the genetic engineering of this organism. Thus, developing an efficient and convenient method for genome editing is imperative. An efficient genome editing method for was developed using an electroporation-based CRISPR-Cas9 technique. In our study, the electroporation efficiency of was found to be limited by its restriction-modification (RM) systems. By searching the putative RM systems in H16 using REBASE database and comparing with that in MG1655, five putative restriction endonuclease genes which are related to the RM systems in were predicated and disrupted. It was found that deletion of and - increased the electroporation efficiency 1658 and 4 times, respectively. Fructose was found to reduce the leaky expression of the arabinose-inducible pBAD promoter, which was used to optimize the expression of , enabling genome editing via homologous recombination based on CRISPR-Cas9 in . A total of five genes were edited with efficiencies ranging from 78.3 to 100%. The CRISPR-Cpf1 system and the non-homologous end joining mechanism were also investigated, but failed to yield edited strains. We present the first genome editing method for using an electroporation-based CRISPR-Cas9 approach, which significantly increased the efficiency and decreased time to manipulate this facultative chemolithoautotrophic microbe. The novel technique will facilitate more advanced researches and applications of for PHA production and CO conversion.