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 sgRNA-ncRNA 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 Cells -- In Vitro Assays for Comparing the Specificity of First- and Next-Generation CRISPR/Cas9 Systems -- Profiling Genome-Wide Specificity of CRISPR-Cas9 Using Digenome-Seq -- Detection of CRISPR/Cas9-Generated Off-Target Effect by Integration-Defective Lentiviral Vector -- Genome-Wide CRISPR Off-Target DNA Break Detection by the BLISS Method.

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. Authoritative and essential, CRISPR Guide RNA Design: Methods and Protocols provides 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.