title: Characterizing Metal-Dependent Nucleases of CRISPR-Cas Prokaryotic Adaptive Immunity Systems.
abstract: 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).
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, 14853, USA.
authors.forename: Ki H
authors.initials: KH
authors.lastname: Nam
authors.forename: Matthew P
authors.initials: MP
authors.lastname: DeLisa
authors.forename: Ailong
authors.initials: A
authors.lastname: Ke
mesh_terms: D056704:Adaptive Immunity; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics / Q000276:immunology*; D064113:CRISPR-Cas Systems / Q000276:immunology; D002151:Calorimetry; D004720:Endonucleases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D008670:Metals; D004720:Endonucleases