CRISPR Methods and Protocols

This volume presents a list of cutting-edge protocols for the study of CRISPR-Cas defense systems and their applications at the genomic, genetic, biochemical and structural levels. CRISPR: Methods and Protocols guides readers through techniques that have been developed specifically for the analysis of CRISPR-Cas and techniques adapted from standard protocols of DNA, RNA and protein biology. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, CRISPR: Methods and Protocols provides a broad list of tools and techniques to study the interdisciplinary aspects of the prokaryotic CRISPR-Cas defense systems.

Investigating CRISPR RNA Biogenesis and Function using RNA-seq.- In vitro Co-reconstitution of Cas Protein Complexes.- Analysis of CRISPR pre-crRNA Cleavage.- Annotation and Classification of CRISPR-Cas Systems.- Computational Detection of CRISPR/crRNA Targets.- High-throughput CRISPR typing of Mycobacterium tuberculosis Complex and Salmonella enterica Serotype Typhimurium.- Spacer-based Macroarrays for CRISPR Genotyping.- Analysis of crRNA using Liquid Chromatography Electrospray Ionisation Mass Spectrometry (LC ESI MS).- Rapid Multiplex Creation of Escherichia coli Strains Capable of Interfering with Phage Infection through CRISPR.- Exploring CRISPR Interference by Transformation with Plasmid Mixtures: Identification Of Target Interference Motifs in Escherichia coli.- Electrophoretic Mobility Shift Assay of DNA and CRISPR-Cas Ribonucleoprotein Complexes.- Expression and Purification of the CMR (Type III-B) Complex in Sulfolobus solfataricus.- Procedures for Generating CRISPR Mutants With Novel Spacers Acquired from Viruses Or Plasmids .- Archaeal Viruses of the Sulfolobales: Isolation, Infection, and CRISPR Spacer Acquisition.- Using the CRISPR-Cas system to Positively Select Mutants In Genes Essential for its Functionl.- Analysis of Nuclease Activity of Cas1 Proteins Against Complex DNA Substrates.- Characterizing Metal-Dependent Nucleases of CRISPR-Cas prokaryotic Adaptive Immunity Systems.- Cas3 Nuclease-Helicase Activity Assays.- Chemical and Enzymatic Footprint Analyses of R-loop Formation by Cascade-crRNA Complex.- Creation andAnalysis of a Virome: Using CRISPR Spacers.- Targeted Mutagenesis in Zebrafish using CRISPR RNA-guided Nucleases.- Precise Genome Editing of Drosophila with CRISPR RNA-guided Cas9.- Targeted Transcriptional Repression in Bacteria using CRISPR Interference (CRISPRi).