Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System

Abstract

ABSTRACTAn efficient genome-scale editing tool is required for construction of industrially useful microbes. We describe a targeted, continual multigene editing strategy that was applied to theEscherichia coligenome by using theStreptococcus pyogenestype II CRISPR-Cas9 system to realize a variety of precise genome modifications, including gene deletion and insertion, with a highest efficiency of 100%, which was able to achieve simultaneous multigene editing of up to three targets. The system also demonstrated successful targeted chromosomal deletions inTatumella citrea, another species of theEnterobacteriaceae, with highest efficiency of 100%.