The article by Ann Ran, Patrick Hsu, Jason Wright, Vineeta Agarwala, David Scott, and Feng Zhang provides a comprehensive guide to using the CRISPR-Cas9 system for genome engineering in eukaryotic cells. The authors describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR), as well as methods for generating modified cell lines for downstream functional studies. To minimize off-target cleavage, they introduce a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. The protocol includes guidelines for selecting target sites, evaluating cleavage efficiency, and analyzing off-target activity. The process can be completed within 1–2 weeks for gene modifications and 2–3 weeks for the derivation of modified clonal cell lines. The article also compares the Cas9 system with other genome editing technologies, highlighting its advantages in terms of ease of customization, targeting efficiency, and multiplexing capacity. Additionally, it discusses the limitations of the Cas9 system, such as the requirement for a specific PAM sequence and the potential for off-target mutagenesis. The authors provide detailed experimental procedures, including the design of targeting components, construction of sgRNA expression constructs, functional validation of sgRNAs, and functional testing of modified cell lines.The article by Ann Ran, Patrick Hsu, Jason Wright, Vineeta Agarwala, David Scott, and Feng Zhang provides a comprehensive guide to using the CRISPR-Cas9 system for genome engineering in eukaryotic cells. The authors describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR), as well as methods for generating modified cell lines for downstream functional studies. To minimize off-target cleavage, they introduce a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. The protocol includes guidelines for selecting target sites, evaluating cleavage efficiency, and analyzing off-target activity. The process can be completed within 1–2 weeks for gene modifications and 2–3 weeks for the derivation of modified clonal cell lines. The article also compares the Cas9 system with other genome editing technologies, highlighting its advantages in terms of ease of customization, targeting efficiency, and multiplexing capacity. Additionally, it discusses the limitations of the Cas9 system, such as the requirement for a specific PAM sequence and the potential for off-target mutagenesis. The authors provide detailed experimental procedures, including the design of targeting components, construction of sgRNA expression constructs, functional validation of sgRNAs, and functional testing of modified cell lines.