Managing the SOS Response for Enhanced CRISPR-Cas-Based Recombineering in E. coli through Transient Inhibition of Host RecA Activity

Phage-derived “recombineering” methods are utilized for bacterial genome editing. 15 Recombineering results in a heterogeneous population of modified and unmodified 16 chromosomes and therefore selection methods, such as CRISPR-Cas9, are required to select 17 for edited clones. Cells can evade CRISPR-Cas-induced cell death through recA-mediated 18 induction of the SOS response. The SOS response increases RecA dependent repair as well 19 mutation rates through the induction of the umuDC error prone polymerase. As a result, 20 CRISPR-Cas selection is more efficient in recA mutants. We report an approach to inhibiting 21 the SOS response and RecA activity through the expression of a mutant dominant negative 22 form of RecA, which incorporates into wild type RecA filaments and inhibits activity. Using 23 a plasmid-based system in which Cas9 and recA mutants are co-expressed, we can achieve 24 increased efficiency and consistency of CRISPR-Cas9-mediated selection and 25 recombineering in E. coli, while reducing the induction of the SOS response. To date, this 26 approach has been shown to be independent of recA genotype and host strain lineage. Using 27 this system, we demonstrate increased CRISPR-Cas selection efficacy with over 10,000 28 guides covering the E. coli chromosome. The use of dominant negative RecA or homologs 29 may be of broad use in bacterial CRISPR-Cas-based genome editing where the SOS 30 pathways are present.