CRISPR Guide RNA Design: Methods and Protocols [Hardcover]

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 successful Methods in Molecular Biology series, 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. Cloud-Based Design of Short Guide RNA (sgRNA) Libraries for CRISPR Experiments.- Web-Based CRISPR Toolkits: Cas-OFFinder, Cas-Designer, and Cas-Analyzer.- Chemical Modification of Guide RNAs for Improved CRISPR Activity in CD34+ Human Hematopoietic Stem and Progenitor Cells.- Gene Disruption Using Chemically-Modified CRISPR-Cpf1 RNA.- Split-and-Click sgRNA.- Chimeric DNA-RNA Guide RNA Designs.- Harnessing tRNA for Processing Ability and Promoter Activity.- Targeting Noncoding RNA Domains to Genomic Loci with CRISPR-Display: Guidelines for Designing, Building, and Testing sgRNAncRNA Expression Constructs.- Controlling the Activity of CRISPR Transcriptional Regulators with Inducible sgRNAs.- Gene Manipulation Using Fusion Guide RNAs for Cas9 and Cas12a.- Methods for Measuring CRISPR/Cas9 DNA Cleavage in Cellæ