This article describes the use of the CRISPR-Cas9 system for genome engineering in eukaryotic cells. The CRISPR-Cas9 system is a powerful tool for targeted genome modification, allowing for precise editing of DNA sequences. The system uses a guide RNA (gRNA) to direct the Cas9 nuclease to a specific location in the genome, where it makes a double-strand break. This break can be repaired through two main pathways: nonhomologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ can lead to insertions or deletions (indels) in the genome, while HDR can be used to introduce specific genetic changes. The article outlines the design of gRNAs, the construction of sgRNA expression plasmids, and the use of these plasmids for genome editing in mammalian cells. It also describes the use of the Cas9 nickase mutant, which can nick DNA rather than cleave it, leading to more precise editing. The protocol provides 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 clonal cell line derivation. The CRISPR-Cas9 system is compared to other genome editing technologies such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). It is noted that CRISPR-Cas9 is easier to customize, more efficient, and allows for multiplex genome editing. The system has been used to generate transgenic mice and to edit organisms that are otherwise genetically intractable. The article also describes the experimental design, including the selection of target sequences, the construction of repair templates, and the isolation of clonal cell lines. It provides detailed protocols for the preparation of sgRNA expression constructs, the transfection of cells, and the functional validation of sgRNAs. The article includes information on the materials, reagents, and equipment needed for the experiments, as well as the procedures for genotyping and functional testing. The CRISPR-Cas9 system is a versatile and powerful tool for genome engineering, with applications in basic science, medicine, and biotechnology.This article describes the use of the CRISPR-Cas9 system for genome engineering in eukaryotic cells. The CRISPR-Cas9 system is a powerful tool for targeted genome modification, allowing for precise editing of DNA sequences. The system uses a guide RNA (gRNA) to direct the Cas9 nuclease to a specific location in the genome, where it makes a double-strand break. This break can be repaired through two main pathways: nonhomologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ can lead to insertions or deletions (indels) in the genome, while HDR can be used to introduce specific genetic changes. The article outlines the design of gRNAs, the construction of sgRNA expression plasmids, and the use of these plasmids for genome editing in mammalian cells. It also describes the use of the Cas9 nickase mutant, which can nick DNA rather than cleave it, leading to more precise editing. The protocol provides 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 clonal cell line derivation. The CRISPR-Cas9 system is compared to other genome editing technologies such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). It is noted that CRISPR-Cas9 is easier to customize, more efficient, and allows for multiplex genome editing. The system has been used to generate transgenic mice and to edit organisms that are otherwise genetically intractable. The article also describes the experimental design, including the selection of target sequences, the construction of repair templates, and the isolation of clonal cell lines. It provides detailed protocols for the preparation of sgRNA expression constructs, the transfection of cells, and the functional validation of sgRNAs. The article includes information on the materials, reagents, and equipment needed for the experiments, as well as the procedures for genotyping and functional testing. The CRISPR-Cas9 system is a versatile and powerful tool for genome engineering, with applications in basic science, medicine, and biotechnology.