Genome editing relies on introduction of a double strand break at a target locus using “designer nucleases” that selectively target one site in the genome. The cell repairs the double strand break through either non-homologous end joining (NHEJ), creating indel mutations, or homology-directed repair (HDR), resulting in knockin modification near the nuclease cutting site. Potential nuclease platforms include zinc finger nucleases and transcription activator-like effector nucleases (TALENs)6. However, designing these nucleases is labor intensive and not readily multiplexed. More recently, the CRISPR/Cas9 nuclease has emerged as a powerful and malleable tool to introduce targeted double strand breaks6. Unlike zinc finger nucleases and TALENs, Cas9 specificity is determined by Watson-Crick base pairing between an engineered guide RNA (gRNA) and the target site7,8. As a result, Cas9 targeting is easily achieved by synthesizing the desired gRNA.
