Microbial genome editing

In an absence of efficient non-homologous end joining (NHEJ) repair mechanisms in the majority of microbes, double stranded DNA break (DSDB) typically leads to cell death. In methods of microbial gene editing using plasmid transformation, both homologous recombination and Cas9 site-specific gene editing events can be used together. Single or multiple plasmid approaches are used. In a method of counter-selection of microbes for a desired genetic change, a two-phase approach is used whereby a switch is made from a higher growth temperature phase favouring homologous recombination (HR) - as opposed to a Cas9 site-directed nuclease activity- to a lower growth temperature phase at which the Cas9 site directed nuclease activity takes place. This has the effect whereby the Cas9 site-directed nuclease activity has counter selecting activity, removing microbes which do not have a desired modification introduced beforehand by HR. The population of microbes surviving after the temperature switch counter selection is thereby enhanced for the desired modification.