GUIDE-Seq is a method for genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. It enables the detection of DNA double-stranded breaks (DSBs) introduced by CRISPR RNA-guided nucleases (RGNs) and other nucleases. The method involves capturing double-stranded oligodeoxynucleotides into breaks and mapping their integration sites in genomic DNA. Application of GUIDE-Seq to thirteen RGNs in two human cell lines revealed wide variability in RGN off-target activities and unappreciated characteristics of off-target sequences. The majority of identified sites were not detected by existing computational methods or ChIP-Seq. GUIDE-Seq also identified RGN-independent genomic breakpoint 'hotspots'. Finally, GUIDE-Seq revealed that truncated guide RNAs exhibit substantially reduced RGN-induced off-target DSBs. Our experiments define the most rigorous framework for genome-wide identification of RGN off-target effects to date and provide a method for evaluating the safety of these nucleases prior to clinical use. CRISPR-Cas RGNs are robust genome-editing reagents with a broad range of research and potential clinical applications. However, therapeutic use of RGNs in humans will require a comprehensive knowledge of their off-target effects to minimize the risk of deleterious outcomes. DNA cleavage by S. pyogenes Cas9 nuclease is directed by a programmable ~100 nt guide RNA (gRNA). Targeting is mediated by 17-20 nts at the gRNA 5'-end, which are complementary to a "protospacer" DNA site that lies next to a protospacer adjacent motif (PAM) of the form 5'-NGG. Repair of blunt-ended Cas9-induced DNA double-stranded breaks (DSBs) within the protospacer by non-homologous end-joining (NHEJ) can induce variable-length insertion/deletion mutations (indels). Our group and others have previously shown that unintended RGN-induced indels can occur at off-target cleavage sites that differ by as many as five positions within the protospacer or that harbor alternative PAM sequences. In addition, chromosomal translocations can result from joining of on- and off-target RGN-induced cleavage events. For clinical applications, identification of even low frequency alterations will be critically important because ex vivo and in vivo therapeutic strategies using RGNs are expected to require the modification of very large cell populations. The induction of oncogenic transformation in even a rare subset of cell clones (e.g., inactivating mutations of a tumor suppressor gene or formation of a tumorigenic chromosomal translocation) is of particular concern because such an alteration could lead to unfavorable clinical outcomes. The identification of indels or higher-order rearrangements that can occur anywhere in the genome is a challenge that is not easily addressed and sensitive methods for unbiased, genome-wide identificationGUIDE-Seq is a method for genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. It enables the detection of DNA double-stranded breaks (DSBs) introduced by CRISPR RNA-guided nucleases (RGNs) and other nucleases. The method involves capturing double-stranded oligodeoxynucleotides into breaks and mapping their integration sites in genomic DNA. Application of GUIDE-Seq to thirteen RGNs in two human cell lines revealed wide variability in RGN off-target activities and unappreciated characteristics of off-target sequences. The majority of identified sites were not detected by existing computational methods or ChIP-Seq. GUIDE-Seq also identified RGN-independent genomic breakpoint 'hotspots'. Finally, GUIDE-Seq revealed that truncated guide RNAs exhibit substantially reduced RGN-induced off-target DSBs. Our experiments define the most rigorous framework for genome-wide identification of RGN off-target effects to date and provide a method for evaluating the safety of these nucleases prior to clinical use. CRISPR-Cas RGNs are robust genome-editing reagents with a broad range of research and potential clinical applications. However, therapeutic use of RGNs in humans will require a comprehensive knowledge of their off-target effects to minimize the risk of deleterious outcomes. DNA cleavage by S. pyogenes Cas9 nuclease is directed by a programmable ~100 nt guide RNA (gRNA). Targeting is mediated by 17-20 nts at the gRNA 5'-end, which are complementary to a "protospacer" DNA site that lies next to a protospacer adjacent motif (PAM) of the form 5'-NGG. Repair of blunt-ended Cas9-induced DNA double-stranded breaks (DSBs) within the protospacer by non-homologous end-joining (NHEJ) can induce variable-length insertion/deletion mutations (indels). Our group and others have previously shown that unintended RGN-induced indels can occur at off-target cleavage sites that differ by as many as five positions within the protospacer or that harbor alternative PAM sequences. In addition, chromosomal translocations can result from joining of on- and off-target RGN-induced cleavage events. For clinical applications, identification of even low frequency alterations will be critically important because ex vivo and in vivo therapeutic strategies using RGNs are expected to require the modification of very large cell populations. The induction of oncogenic transformation in even a rare subset of cell clones (e.g., inactivating mutations of a tumor suppressor gene or formation of a tumorigenic chromosomal translocation) is of particular concern because such an alteration could lead to unfavorable clinical outcomes. The identification of indels or higher-order rearrangements that can occur anywhere in the genome is a challenge that is not easily addressed and sensitive methods for unbiased, genome-wide identification