Single-Molecule Observation of Viral DNA Targeting by CRISPR/Cas Immune Systems

Abstract

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). An adaptive immune response is triggered during subsequent infections by CRISPR-derived RNAs (crRNAs), which function together with CRISPR-associated (Cas) proteins to identify and destroy complementary viral DNA sequences. DNA targeting by crRNA/Cas ribonucleoprotein surveillance complexes proceeds via RNA-DNA base-pairing interactions and requires melting of the double-stranded substrate, as well as protein-mediated recognition of a proximal DNA sequence motif to discriminate self from non-self. How these complexes find short target sequences within the larger context of genomic DNA is unknown, and prior studies have been limited by their use of oligonucleotide substrates and dependence on bulk electrophoretic mobility shift assays that obscure all dynamics of the search process. To gain deeper insights into this critical step of CRISPR/Cas based immunity, we are using fluorescence microscopy to visualize single crRNA/Cas complexes on nanofabricated curtains of viral DNA in real-time. These experiments enable direct observation of the kinetics and position-dependence of DNA binding, and have revealed the mechanism by which phylogenetically distinct surveillance complexes use sequence information in the crRNA to locate complementary DNA targets. Ongoing work is aimed at understanding how this recognition event promotes destruction of viral DNA by dedicated Cas nucleases.