Full Description
This detailed volume guides readers through strategic planning and user-friendly guidelines in order to select the most suitable CRISPR-Cas system and target sites with high activity and specificity. Methods covering CRISPR gRNA design, CRISPR delivery, CRISPR activity quantification (indel quantification), and examples of applying CRISPR gene editing in human pluripotent stem cells, primary cells, gene therapy, and genetic screening are included. 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 laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and invaluable, CRISPR Gene Editing: Methods and Protocols will assist undergraduates, graduates, and researchers with detailed guidelines and methods for the vitally important CRISPR gene editing field.
Chapter 3 is available open access under a CC BY 4.0 license via link.springer.com.
Contents
CRISPR-gRNA Design.- Tracking CRISPR's Footprints.- Rapid Quantitative Evaluation of CRISPR Genome Editing by TIDE and TIDER.- Fast and Quantitative Identification of Ex Vivo Precise Genome Targeting-Induced Indel Events by IDAA.- Functional Evaluation of CRISPR Activity by the Dual-Fluorescent Surrogate System: C-Check.- CRISPR-Cas9 Delivery by Artificial Virus (RRPHC).- Production and Validation of Lentiviral Vectors for CRISPR/Cas9 Delivery.- Rapid and Simple Screening of CRISPR Guide RNAs (gRNAs) in Cultured Cells Using Adeno-Associated Viral (AAV) Vectors.- Electroporation-Based CRISPR/Cas9 Gene Editing Using Cas9 Protein and Chemically Modified sgRNAs.- Efficient Gene Editing of Human Induced Pluripotent Stem Cells Using CRISPR/Cas9.- Editing the Genome of Human Induced Pluripotent Stem Cells Using CRISPR/Cas9 Ribonucleoprotein Complexes.- Conditional Gene Knockout in Human Cells with Inducible CRISPR/Cas9.- CRISPR/Cas9 as a Genome Editing Tool for Targeted Gene Integration in CHO Cells.- Rapid and Efficient Gene Deletion by CRISPR/Cas9.- Genome Editing in Mice.- CRISPR/Cas9-Mediated Gene Tagging: A Step-by-Step Protocol.- Gene Editing in Primary Cells of Cattle and Pig.- Toward In Vivo Gene Therapy Using CRISPR.- CRISPR Gene Therapy of the Eye: Targeted Knockout of Vegfa in Mouse Retina by Lentiviral Delivery.- In Vivo Editing of the Adult Mouse Liver Using CRISPR/Cas9 and Hydrodynamic Tail Vein Injection.- CRISPR-Based Lentiviral Knockout Libraries for Functional Genomic Screening and Identification of Phenotype-Related Genes.
