A CRISPR-based system using RNA-guided nucleases was developed for efficient in vivo genome editing. The study demonstrates that this system can induce targeted genetic modifications in zebrafish embryos with efficiencies comparable to those achieved using zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). RNA-guided nucleases robustly enabled genome editing at 9 of 11 different sites tested, including two for which TALENs previously failed to induce alterations. These results show that programmable CRISPR/Cas systems provide a simple, rapid, and highly scalable method for altering genes in vivo, opening the door to using RNA-guided nucleases for genome editing in a wide range of organisms. The CRISPR/Cas system uses clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins to provide acquired resistance to invading viruses and plasmids. The type II CRISPR/Cas system relies on uptake of foreign DNA fragments into CRISPR loci and subsequent transcription and processing of these CRISPR repeat-spacer arrays into short CRISPR RNAs (crRNAs), which in turn anneal to a trans-activating crRNA (tracrRNA) and direct sequence-specific silencing of foreign nucleic acid by Cas proteins. Recent in vitro studies have shown that a single synthetic guide RNA (gRNA), consisting of a fusion of crRNA and tracrRNA, can direct Cas9-mediated cleavage of target DNA. The study explored the abilities of customizable gRNAs and Cas9 nuclease to efficiently modify endogenous genes in vivo in zebrafish embryos and showed that this system provides a rapid and robust alternative to ZFNs and TALENs for performing genome editing in whole organisms. The researchers constructed expression vectors that enable T7 RNA polymerase-mediated production of a capped, poly-adenylated mRNA encoding the monomeric Cas9 nuclease and of a customizable gRNA bearing 20 nucleotides of sequence complementary to a target site. They observed robust induction of targeted insertion/deletion mutations (indels) at all concentrations of RNAs tested and in nearly all individual embryos tested. The study also demonstrated that the gRNA/Cas9 system is robust and can target a wide range of sequences. The researchers constructed ten additional gRNAs targeting another sequence in the fh gene and sites in nine additional endogenous genes. They found that for eight of the ten sites they targeted, co-injection of gRNA with Cas9-encoding mRNA induced high frequencies of targeted indels at these sites in all individual embryos tested. The mutation rates observed were comparable to those previously observed at targets in these same genes using ZFNs and/or TALENs. The study also highlights the advantages of the gRNA/Cas9 system over ZFNs and TALENs. Only one customized gRNAA CRISPR-based system using RNA-guided nucleases was developed for efficient in vivo genome editing. The study demonstrates that this system can induce targeted genetic modifications in zebrafish embryos with efficiencies comparable to those achieved using zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). RNA-guided nucleases robustly enabled genome editing at 9 of 11 different sites tested, including two for which TALENs previously failed to induce alterations. These results show that programmable CRISPR/Cas systems provide a simple, rapid, and highly scalable method for altering genes in vivo, opening the door to using RNA-guided nucleases for genome editing in a wide range of organisms. The CRISPR/Cas system uses clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins to provide acquired resistance to invading viruses and plasmids. The type II CRISPR/Cas system relies on uptake of foreign DNA fragments into CRISPR loci and subsequent transcription and processing of these CRISPR repeat-spacer arrays into short CRISPR RNAs (crRNAs), which in turn anneal to a trans-activating crRNA (tracrRNA) and direct sequence-specific silencing of foreign nucleic acid by Cas proteins. Recent in vitro studies have shown that a single synthetic guide RNA (gRNA), consisting of a fusion of crRNA and tracrRNA, can direct Cas9-mediated cleavage of target DNA. The study explored the abilities of customizable gRNAs and Cas9 nuclease to efficiently modify endogenous genes in vivo in zebrafish embryos and showed that this system provides a rapid and robust alternative to ZFNs and TALENs for performing genome editing in whole organisms. The researchers constructed expression vectors that enable T7 RNA polymerase-mediated production of a capped, poly-adenylated mRNA encoding the monomeric Cas9 nuclease and of a customizable gRNA bearing 20 nucleotides of sequence complementary to a target site. They observed robust induction of targeted insertion/deletion mutations (indels) at all concentrations of RNAs tested and in nearly all individual embryos tested. The study also demonstrated that the gRNA/Cas9 system is robust and can target a wide range of sequences. The researchers constructed ten additional gRNAs targeting another sequence in the fh gene and sites in nine additional endogenous genes. They found that for eight of the ten sites they targeted, co-injection of gRNA with Cas9-encoding mRNA induced high frequencies of targeted indels at these sites in all individual embryos tested. The mutation rates observed were comparable to those previously observed at targets in these same genes using ZFNs and/or TALENs. The study also highlights the advantages of the gRNA/Cas9 system over ZFNs and TALENs. Only one customized gRNA