CRISPR pp 265-276 | Cite as

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

Part of the Methods in Molecular Biology book series (MIMB, volume 1311)

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).

Key words

Cas2 CRISPR/Cas DNase Isothermal titration calorimetry (ITC) Microbial adaptation Nuclease 

Notes

Acknowledgements

This work was supported by the National Institutes of Health Grant GM-102543 (to A.K.).

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Molecular Biology and GeneticsCornell UniversityIthacaUSA
  2. 2.School of Chemical and Biomolecular EngineeringCornell UniversityIthacaUSA

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