The Impact of Cellular Genome Maintenance Proteins on Primed CRISPR Adaptation By the Escherichia coli Type I-E CRISPR-Cas System

Primed CRISPR adaptation by the type I-E CRISPR-Cas system of Escherichia coli is coupled with CRISPR interference and directed by the protospacer targeting. During this process, short DNA fragments from the priming area are incorporated into the CRISPR array to become new spacers, with efficiency of selection gradually falling with a growing distance from the protospacer. However, the exact mechanism of spacer choice and insertion and the participants of these reactions in vivo are not yet fully determined. In this study, we use previously described self-targeting model to establish the impact of several proteins providing bacterial genome stability in primed CRISPR adaptation. By introducing single or double knockout mutations of the corresponding genes and performing high-throughput analysis of acquired spacers, we compare the efficiency and patterns of CRISPR adaptation. Based on the results, the distinct roles of RecBCD, SbcCD, SbcB and RecJ proteins in different steps of primed CRISPR adaptation are proposed, suggesting these proteins to alter the processivity of priming complex or pre-spacer maturation and insertion.