Genome engineering in Escherichia coli is used for the construction of complex biological machines, enabling cell-based manufacturing, novel drugs, and more. Currently, bacterial chromosomal engineering utilizes phage-derived “recombineering” methods to integrate foreign DNA, resulting in deletions, insertions, and substitutions. Recombineering is reliant on the replication fork to integrate donor DNA as an Okazaki fragment. However, this results in a heterogeneous population of modified and unmodified chromosomes. Selection methods are therefore required to kill cells containing unmodified chromosomes. CRISPR/Cas9 has been adopted as a selection tool for its ability to cause cell death by introducing targeted double strand breaks (DSBs). Cas9 could theoretically be versatile enough to target multiple sites at once, a requirement for multiplex genome engineering. However, recent work has shown that cells can evade cell death through the recA-mediated SOS repair pathway and sister chromosomes. CRISPR/Cas9 selection is therefore more efficient in recA-deficient strains. One way of inhibiting recA activity is through the expression of a mutant co-dominant negative form of recA. These recA mutants bind with recA, forming filaments that lack activity. In order to improve recombineering efficiencies, we have developed a plasmid in which Cas9 and recA mutants are co-expressed resulting in the increased efficiency of CRISPR/Cas9-mediated recombineering in E. coli.

Using co-dominant negative recA to enhance CRISPR/Cas9-mediated recombineering

253rd National Meeting (2017)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.