This review discusses the use of CRISPR-Cas9 for high-throughput functional genomics. CRISPR-Cas9, an RNA-guided nuclease, enables targeted modification of DNA sequences. It has been combined with genome-scale guide RNA libraries for unbiased, phenotypic screening. The review outlines recent advances in using Cas9 for genome-scale screens, including knockout approaches and strategies for modulating transcriptional activity. It compares these methods with RNA interference (RNAi) screening and discusses practical aspects of screen design, future applications, and challenges. Forward genetic screens are 'phenotype-to-genotype' approaches that involve modifying or modulating the expression of many genes, selecting for cells or organisms with a phenotype of interest, and then characterizing the mutations that result in those phenotypic changes. These screens have been used in model organisms such as yeast, flies, plants, zebrafish, nematodes, and rodents. However, a major shortcoming of DNA-mutagen-based screens is that the causal mutations in the selected clones are initially unknown. These challenges can now be more easily addressed by mapping mutations using next-generation sequencing and by replacing chemical mutagens with viruses and transposons. An additional limitation of random mutagenesis approaches is that the resulting mutants are typically heterozygotes, which can mask recessive phenotypes. In model organisms, homozygosity can be achieved by intercrossing progeny derived from the initial heterozygous mutant. In mammalian cell culture, recessive screens have been limited to near-haploid cell lines or to cell lines deficient in Bloom helicase. Over the past decade, forward genetic screens have been revolutionized by the development of tools that use the RNA interference (RNAi) pathway for gene knockdown. RNAi is a conserved endogenous pathway that facilitates design and scalability of tools. Several RNAi reagents have been developed, including long double-stranded RNA, synthetic small interfering RNA, short hairpin RNA, and shRNAs embedded in microRNA precursors. Screens using RNAi tools have provided a wealth of information on gene function, but their utility has been hindered by incomplete gene knockdown and extensive off-target activity. Sequence-specific programmable nucleases have emerged as an exciting new genetic perturbation system that enables targeted modification of the DNA sequence itself. The RNA-guided endonuclease Cas9 provides a convenient system for achieving targeted mutagenesis in eukaryotic cells. Cas9 is targeted to specific genomic loci via a guide RNA, which recognizes the target DNA through Watson–Crick base pairing. Therefore, Cas9 combines the permanently mutagenic nature of classical mutagens with the programmability of RNAi. In this review, we discuss recent Cas9-based functional genetic screening tools, including genome-wide knockout approaches and related strategies using modified forms of Cas9 to cause gene knockdown or transcriptional activation in a non-mutagenThis review discusses the use of CRISPR-Cas9 for high-throughput functional genomics. CRISPR-Cas9, an RNA-guided nuclease, enables targeted modification of DNA sequences. It has been combined with genome-scale guide RNA libraries for unbiased, phenotypic screening. The review outlines recent advances in using Cas9 for genome-scale screens, including knockout approaches and strategies for modulating transcriptional activity. It compares these methods with RNA interference (RNAi) screening and discusses practical aspects of screen design, future applications, and challenges. Forward genetic screens are 'phenotype-to-genotype' approaches that involve modifying or modulating the expression of many genes, selecting for cells or organisms with a phenotype of interest, and then characterizing the mutations that result in those phenotypic changes. These screens have been used in model organisms such as yeast, flies, plants, zebrafish, nematodes, and rodents. However, a major shortcoming of DNA-mutagen-based screens is that the causal mutations in the selected clones are initially unknown. These challenges can now be more easily addressed by mapping mutations using next-generation sequencing and by replacing chemical mutagens with viruses and transposons. An additional limitation of random mutagenesis approaches is that the resulting mutants are typically heterozygotes, which can mask recessive phenotypes. In model organisms, homozygosity can be achieved by intercrossing progeny derived from the initial heterozygous mutant. In mammalian cell culture, recessive screens have been limited to near-haploid cell lines or to cell lines deficient in Bloom helicase. Over the past decade, forward genetic screens have been revolutionized by the development of tools that use the RNA interference (RNAi) pathway for gene knockdown. RNAi is a conserved endogenous pathway that facilitates design and scalability of tools. Several RNAi reagents have been developed, including long double-stranded RNA, synthetic small interfering RNA, short hairpin RNA, and shRNAs embedded in microRNA precursors. Screens using RNAi tools have provided a wealth of information on gene function, but their utility has been hindered by incomplete gene knockdown and extensive off-target activity. Sequence-specific programmable nucleases have emerged as an exciting new genetic perturbation system that enables targeted modification of the DNA sequence itself. The RNA-guided endonuclease Cas9 provides a convenient system for achieving targeted mutagenesis in eukaryotic cells. Cas9 is targeted to specific genomic loci via a guide RNA, which recognizes the target DNA through Watson–Crick base pairing. Therefore, Cas9 combines the permanently mutagenic nature of classical mutagens with the programmability of RNAi. In this review, we discuss recent Cas9-based functional genetic screening tools, including genome-wide knockout approaches and related strategies using modified forms of Cas9 to cause gene knockdown or transcriptional activation in a non-mutagen