Detection of Recbcd-Generated DNA Products in Escherichia coli Cells Undergoing CRISPR Interference

CRISPR interference by the Escherichia coli subtype I-E CRISPR-Cas system is an efficient process of target degradation by Cas3 helicase/nuclease. In plasmid-based experiments, CRISPR interference leads to full degradation of the target. However, small size of plasmids doesn’t allow to detect the maximal distance to which degradation can occur. We previously developed a self-targeting system where a single chromosomally located protospacer is recognized by the type I-E CRISPR interference machinery. Using this system we show that CRISPR interference proceeds to both directions, upstream and downstream from the targeted protospacer, leading to degradation of up to 300 kb of genomic DNA.

DNA with a double-stranded gap is a substrate for RecBCD-mediated DNA repair in E. coli. Upon binding to a DNA end, RecBCD helicase/nuclease translocates along DNA until it reaches 8-nt sequence termed Chi-site. The recognition of Chi results in nicking of 3’-terminated strand and loading of RecA recombinase on a nascent 3’end. Despite decades of studying, enzymatic behavior of RecBCD before it pauses at Chi is not understood. In vitro RecBCD cleaves both DNA strands producing fragments with a length substantially varying under different reaction conditions. No RecBCD-generated products have been detected in vivo yet. We show here that in the self-targeting system degradation of genomic DNA started by CRISPR interference machinery is further continued by RecBCD complex. Using a high-throughput sequencing approach for detecting short DNA fragments, “FragSeq,” we characterize RecBCD-generated products and establish how cleavage pattern depends on a strand and position with respect to Chi-sites in vivo.