The ability to engineer biological systems and organisms holds enormous potential for applications across basic science, medicine and biotechnology. Programmable sequence-specific endonucleases that facilitate precise editing of endogenous genomic loci are now enabling systematic interrogation of genetic elements and causal genetic variations1,2 in a broad range of species, including those that have not previously been genetically tractable3–6. A number of genome editing technologies have emerged in recent years, including zinc-finger nucleases (ZFNs)7–10, transcription activator–like effector nucleases (TALENs)10–17 and the RNA-guided CRISPR-Cas nuclease system18–25. The first two technologies use a strategy of tethering endonuclease catalytic domains to modular DNA-binding proteins for inducing targeted DNA double-stranded breaks (DSBs) at specific genomic loci. By contrast, Cas9 is a nuclease guided by small RNAs through Watson-Crick base pairing with target DNA26–28 (Fig. 1), representing a system that is markedly easier to design, highly specific, efficient and well-suited for high-throughput and multiplexed gene editing for a variety of cell types and organisms.
