Characterizing Metal-Dependent Nucleases of CRISPR-Cas Prokaryotic Adaptive Immunity Systems.

CRISPRs (clustered regularly interspaced short palindromic repeats), together with the nearby CRISPR-associated (cas) operon, constitute a prokaryotic RNA-based adaptive immune system against exogenous genetic elements. Here, we describe nuclease assays that are useful for characterizing the substrate-specific function of CRISPR-associated protein Cas2. We also provide methods for characterizing the stoichiometry and affinity between Cas2 and divalent metal ions using isothermal titration calorimetry (ITC).