Determinants for efficient editing with Cas9-mediated recombineering in Escherichia coli

In E. coli, editing efficiency (EE) with Cas9-mediated recombineering varies across targets due to differences in the level of Cas9:gRNA DNA double-strand break (DSB)-induced cell death. We found that EE with the same gRNA and repair template can also change with target position, cas9 promoter strength, and growth conditions. Incomplete editing, off-target activity, non-targeted mutations, and failure to cleave target DNA even if Cas9 is bound also compromise EE. These effects on EE were gRNA-specific. We propose that differences in the efficiency of Cas9:gRNA-mediated DNA DSBs and differences in rates of dissociation of Cas9:gRNA complexes from target sites account for the observed variations in EE between gRNAs. We show that editing behavior using the same gRNA can be modified by mutating the gRNA spacer, which changes the DNA DSB activity. Finally, we discuss how variable editing with different gRNAs could limit high-throughput applications and provide strategies to overcome these limitations. Overall design: Examination of edited genomic loci for 8 different editing cassettes each with a unique gRNA and homology repair template targeting galK by placing galK at different loci and Cas9 under different promoters. A 20 bp sequence homologous to the sequence upstream of the PAM was used as the gRNA spacer. In our editing cassettes for point mutations, we designed 150 bp homology repair templates (HRTs) centered around the NGG PAM. This template consisted a synonymous mutation to mutate the G in the NGG PAM to provide immunity to subsequent cleavage by Cas9 after editing as well as targeted mutation(s) within 20 bp of the synonymous PAM mutation. This repair homology template was placed in cis with the gRNA, which was expressed under control of the J23119 promoter.