Defense Against Viral Attack: Single-Molecule View on a Bacterial Adaptive Immune System

Abstract

Escherichia coli maintain different strategies to protect the cell against invading foreign DNA. In a recently discovered adaptive immune system, fragments of foreign DNA are integrated into specific loci on the bacterial genome, known as clustered regularly interspaced short palindromic repeats (CRISPR). Short CRISPR-derived RNAs (crRNAs) are incorporated into the CRISPR-associated complex for antiviral defence (Cascade) and guide the complex's search for the DNA of returning invaders, which is targeted for destruction upon binding. Recent studies have shown that Cascade must recognize both a target sequence and an immediately adjacent PAM (protospacer adjacent motif) sequence in order for successful targeting of the foreign DNA. The mechanism and structural dynamics of this target recognition and binding process, however, are not well understood. Here we report a single-molecule FRET (Forster resonance energy transfer) based assay to monitor in real time the target recognition process of Cascade. We observe that Cascade binds target DNAs that vary widely in sequence. Notably, a complementary target sequence with a correct PAM will be completely opened up by Cascade, whereas an identical sequence with a mutated PAM will be only partially opened. In addition, the latter exhibits a weaker affinity to Cascade. A sequence that contains a PAM but lacks a correct target sequence, still exhibits binding, but is not opened. Taken together, these results suggest that the discrimination of self from non-self occurs after promiscuous target binding. Our single-molecule study promises to reveal further mechanistic details of target DNA identification by the CRISPR immune system.