RNA-guided Cas9 nucleases derived from clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have dramatically transformed our ability to edit the genomes of diverse organisms. We believe tools and techniques based on Cas9, a single unifying factor capable of colocalizing RNA, DNA and protein, will grant unprecedented control over cellular organization, regulation and behavior. Here we describe the Cas9 targeting methodology, detail current and prospective engineering advances and suggest potential applications ranging from basic science to the clinic.Bacteria and archaea have evolved adaptive immune defenses termed CRISPR-CRISPRassociated (Cas) systems that use short RNA to direct degradation of foreign nucleic acids 1-3 . Type II CRISPR-Cas systems have been engineered to effect robust RNA-guided genome modifications in multiple eukaryotic systems 4-17 , substantially improving the ease of genome editing and, more recently, genome regulation [18][19][20][21][22][23] . As an RNA-guided dsDNAbinding protein, the Cas9 effector nuclease is the first known example of a programmable unifying factor capable of colocalizing all three types of sequence-defined biological polymers, a capability with tremendous potential for engineering living systems. Here we review the Cas9 targeting methodology, outline key steps toward enhancing the efficacy, specificity and versatility of Cas9-mediated genome editing and regulation, and highlight its transformative potential for basic science, cellular engineering and therapeutics.
Engineering CRISPR-Cas systemsIn bacteria and archaea, CRISPR-Cas systems provide immunity by incorporating fragments of invading phage and plasmid DNA into CRISPR loci and using the corresponding CRISPR RNAs (crRNAs) to guide the degradation of homologous sequences 24 . Each CRISPR locus encodes acquired 'spacers' that are separated by repeat sequences. Transcription of the locus yields a pre-crRNA, which is processed to yield crRNAs consisting of spacer-repeat fragments that guide effector nuclease complexes to cleave