Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have evolved in bacteria and archaea as a defense mechanism to silence foreign nucleic acids of viruses and plasmids. Recent work has shown that bacterial type II CRISPR systems can be adapted to create guide RNAs (gRNAs) capable of directing site-specific DNA cleavage by the Cas9 nuclease in vitro. Here we show that this system can function in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies comparable to those obtained using ZFNs and TALENs for the same genes. 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 demonstrate that programmable CRISPR/Cas systems provide a simple, rapid, and highly scalable method for altering genes in vivo, opening the door to using RNAguided nucleases for genome editing in a wide range of organisms. Bacteria and archaea have evolved an elegant adaptive defense mechanism which uses clustered regularly interspaced short palindromic repeats (CRISPR), together with CRISPRassociated (Cas) proteins, to provide acquired resistance to invading viruses and plasmids 1-3 . The type II CRISPR/Cas system relies on uptake of foreign DNA fragments into CRISPR loci 4 and subsequent transcription and processing of these CRISPR repeatspacer arrays into short CRISPR RNAs (crRNAs) 5 , which in turn anneal to a transactivating crRNA (tracrRNA) and direct sequence-specific silencing of foreign nucleic acid by Cas proteins 5-7
