Escherichia coli

Prokaryotes encode adaptive immune systems called CRISPR-Cas to provide resistance against mobile invaders such as viruses and plasmids. Host immunity is based on incorporation of invader DNA sequences in a memory locus (CRISPR), the formation of guide RNAs from this locus and the degradation of cognate invader DNA (protospacer). Invaders can escape Type I-E CRISPR-Cas immunity in Escherichia coli K12 by making point mutations in the seed region of the protospacer or its adjacent motif (PAM), but hosts quickly restore immunity by integrating new spacers in a positive feedback process called priming. Here, by using a randomized protospacer and PAM library and high-throughput loss assays we provide a detailed analysis of the constraints of direct interference and subsequent priming in E. coli. We have defined a high-resolution genetic map of direct interference by Cascade and Cas3, which includes five positions of the protospacer at 6 nt intervals that tolerate mutations. Importantly, we show that priming is an extremely robust process capable of utilizing degenerate target regions with up to eleven mutations throughout the PAM and protospacer region. Our findings imply that even out-dated spacers containing many mismatches can induce a rapid primed CRISPR response against diversified or related invaders, giving microbes an advantage in the co-evolutionary arms race with their invaders.