The CRISPR-Cas9 system is a powerful tool for precise genome editing in eukaryotic cells. This review describes the use of Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as the generation of modified cell lines for downstream functional studies. The system uses RNA-guided Cas9 nuclease, which can be directed to specific genomic loci by specifying a 20-nt targeting sequence within its guide RNA. To minimize off-target cleavage, a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs is described. This protocol provides guidelines for selecting target sites, evaluating cleavage efficiency, and analyzing off-target activity. Gene modifications can be achieved within 1–2 weeks, and modified clonal cell lines can be derived within 2–3 weeks. The CRISPR-Cas system is a microbial adaptive immune system that uses RNA-guided nucleases to cleave foreign genetic elements. The Type II CRISPR system is one of the best characterized, consisting of the nuclease Cas9, the crRNA array that encodes the guide RNAs, and a required auxiliary trans-activating crRNA (tracrRNA) that facilitates the processing of the crRNA array into discrete units. Each crRNA unit contains a 20-nt guide sequence and a partial direct repeat, where the former directs Cas9 to a 20-bp DNA target via Watson-Crick base pairing. Cas9 promotes genome editing by stimulating a DSB at a target genomic locus. Upon cleavage by Cas9, the target locus typically undergoes one of two major pathways for DNA damage repair: the error-prone NHEJ or the high-fidelity HDR pathway. In the absence of a repair template, DSBs are re-ligated through the NHEJ process, which leaves scars in the form of insertion/deletion (indel) mutations. NHEJ can be harnessed to mediate gene knockouts, as indels occurring within a coding exon can lead to frameshift mutations and premature stop codons. Multiple DSBs can additionally be exploited to mediate larger deletions in the genome. HDR is an alternative major DNA repair pathway. Although HDR typically occurs at lower and substantially more variable frequencies than NHEJ, it can be leveraged to generate precise, defined modifications at a target locus in the presence of an exogenously introduced repair template. The repair template can either be in the form of conventional double-stranded DNA targeting constructs with homology arms flanking the insertion sequence, or single-stranded DNA oligonucleotides (ssODNs). The latter provides an effective and simple method for making small edits in the genome, such as the introduction of single-nucleotide mutations for probing causal genetic variations. The RNA-guided nuclease function of CRThe CRISPR-Cas9 system is a powerful tool for precise genome editing in eukaryotic cells. This review describes the use of Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as the generation of modified cell lines for downstream functional studies. The system uses RNA-guided Cas9 nuclease, which can be directed to specific genomic loci by specifying a 20-nt targeting sequence within its guide RNA. To minimize off-target cleavage, a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs is described. This protocol provides guidelines for selecting target sites, evaluating cleavage efficiency, and analyzing off-target activity. Gene modifications can be achieved within 1–2 weeks, and modified clonal cell lines can be derived within 2–3 weeks. The CRISPR-Cas system is a microbial adaptive immune system that uses RNA-guided nucleases to cleave foreign genetic elements. The Type II CRISPR system is one of the best characterized, consisting of the nuclease Cas9, the crRNA array that encodes the guide RNAs, and a required auxiliary trans-activating crRNA (tracrRNA) that facilitates the processing of the crRNA array into discrete units. Each crRNA unit contains a 20-nt guide sequence and a partial direct repeat, where the former directs Cas9 to a 20-bp DNA target via Watson-Crick base pairing. Cas9 promotes genome editing by stimulating a DSB at a target genomic locus. Upon cleavage by Cas9, the target locus typically undergoes one of two major pathways for DNA damage repair: the error-prone NHEJ or the high-fidelity HDR pathway. In the absence of a repair template, DSBs are re-ligated through the NHEJ process, which leaves scars in the form of insertion/deletion (indel) mutations. NHEJ can be harnessed to mediate gene knockouts, as indels occurring within a coding exon can lead to frameshift mutations and premature stop codons. Multiple DSBs can additionally be exploited to mediate larger deletions in the genome. HDR is an alternative major DNA repair pathway. Although HDR typically occurs at lower and substantially more variable frequencies than NHEJ, it can be leveraged to generate precise, defined modifications at a target locus in the presence of an exogenously introduced repair template. The repair template can either be in the form of conventional double-stranded DNA targeting constructs with homology arms flanking the insertion sequence, or single-stranded DNA oligonucleotides (ssODNs). The latter provides an effective and simple method for making small edits in the genome, such as the introduction of single-nucleotide mutations for probing causal genetic variations. The RNA-guided nuclease function of CR