CRISPR (clustered regularly interspaced short palindromic repeats) RNA and CRISPR-associated genes (Cas) assemble into RNA-guided surveillance complex that targets foreign nucleic acids for destruction. There are two major classes of CRISPR-Cas systems. Class I systems utilize a multi-subunit effector complex called Cascade to recognize and degrade target nucleic acids; whereas, Class II systems employ a single polypeptide for these activities. Here, I present structures of a novel, uncharacterized Type IV surveillance complex from Class I. It has striking similarity to Type III Cmr effector complexes. It is tempting to hypothesize that it targets single-stranded RNA. I also present detailed kinetic characterization of high-fidelity Class II Cas9 variants. These high-fidelity enzymes achieve specificity by altering the kinetic portioning and allowing substrate release prior to the irreversible cleavage reaction.
1. Understand how cryo-EM can be used for structural studies of CRISPR-Cas complexes.
2. Interpret kinetic data of Cas9 complexes.