CRISPR-Cas-based techniques are revolutionizing the way geneticists and molecular biologists modify DNA sequences and modulate gene expression in cells and organisms. This laboratory manual presents step-by-step protocols for applying this cutting-edge technology to any system of interest. Contributors describe approaches for designing guide RNAs that precisely target the gene of interest, delivering CRISPR-Cas reagents into cells (e.g., using AAV vectors), and selecting cells that harbor the desired changes. Strategies for scaling and multiplexing these procedures are also described, as are methods for optimizing CRISPR-Cas systems in common species and cell lines, from yeast to human.