The article provides a comprehensive guide to using the CRISPR-Cas9 system for genome engineering in eukaryotic cells. The authors describe the use of Cas9, guided by small RNAs, to facilitate precise genome editing through nonhomologous end joining (NHEJ) or homology-directed repair (HDR). They detail the design of targeting sequences, construction and validation of single-guide RNAs (sgRNAs), and methods for functional validation of gene modifications. The protocol includes strategies to minimize off-target effects, such as using a double-nicking strategy with Cas9 nickase mutants. The article also covers the selection of target sites, evaluation of cleavage efficiency, and analysis of off-target activity. The authors provide experimental guidelines and computational tools to assist in the design process, emphasizing the ease and efficiency of Cas9-mediated genome editing compared to other technologies like ZFNs and TALENs.The article provides a comprehensive guide to using the CRISPR-Cas9 system for genome engineering in eukaryotic cells. The authors describe the use of Cas9, guided by small RNAs, to facilitate precise genome editing through nonhomologous end joining (NHEJ) or homology-directed repair (HDR). They detail the design of targeting sequences, construction and validation of single-guide RNAs (sgRNAs), and methods for functional validation of gene modifications. The protocol includes strategies to minimize off-target effects, such as using a double-nicking strategy with Cas9 nickase mutants. The article also covers the selection of target sites, evaluation of cleavage efficiency, and analysis of off-target activity. The authors provide experimental guidelines and computational tools to assist in the design process, emphasizing the ease and efficiency of Cas9-mediated genome editing compared to other technologies like ZFNs and TALENs.