Targeting And Degradation Of Viral Dna By The Crispr-Cas System Of Escherichia Coli

Bacteria and archaea maintain a history of viral infections by integrating small fragments of foreign DNA into specialized genomic loci called clustered regularly interspaced short palindromic repeats (CRISPRs). Subsequent infections trigger an adaptive immune response, which relies on both CRISPR RNAs (crRNAs) and CRISPR-associated (Cas) proteins to identify and destroy invading DNA. In E. coli, the crRNA/Cas ribonucleoprotein surveillance complex is referred to as Cascade. Cascade recognizes foreign DNA targets via crRNA-DNA base pairing and subsequently recruits a trans-acting nuclease/helicase, Cas3, for degradation of the targeted DNA. To fully dissect the mechanistic features of target recognition and degradation by Cascade/Cas3, we have used a combination of single molecule and bulk methods. We reveal crucial features in DNA that Cascade senses to locate and recognize complementary target sequences within the larger context of genomic DNA. Further, we uncover the key elements necessary for Cascade-dependent recruitment of Cas3. Finally, by directly visualizing Cas3 as it unwinds and cleaves viral DNA, we characterize the degradation machinery in exceptional detail, providing fundamental insights into the mechanism of CRISPR-based immunity. Importantly, by comparing these results with our recent findings for another CRISPR-Cas system, Cas9 in S. pyogenes, we can begin to understand the evolution of DNA targeting machineries in related RNA-based adaptive immune systems.