This detailed volume focuses on the CRISPR-associated guide RNA and how it can be designed, modified, and validated for a broad repertoire of purposes. Beginning with a section on computational design of target-specific guide RNAs, the book continues by covering chemical modifications to alter guide RNA stability, specificity, and efficiency, as well as to create inducible guide RNAs, append additional functional domains, and express guide RNAs in a conditional manner. It concludes with methods for measuring off-target guide RNA activity. Written for the highly successfulMethods in Molecular Biologyseries, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and essential,CRISPR Guide RNA Design: Methods and Protocolsprovides a comprehensive pipeline for guide RNA design and aims to be an invaluable resource in applying this powerful technology to basic research and therapeutic applications.