The RNA-programmable Cas9 endonuclease cleaves double-stranded DNA at sites complementary to a 20-base-pair guide RNA. The Cas9 system has been used to modify genomes in multiple cells and organisms, demonstrating its potential as a facile genome-engineering tool. We used in vitro selection and high-throughput sequencing to determine the propensity of eight Cas9:guide RNA complexes to cleave each of 10^12 potential off-target DNA sequences. The selection results predicted five off-target sites in the human genome that were confirmed to undergo genome cleavage in HEK293T cells upon expression of one of two Cas9:guide RNA complexes. In contrast to previous models, our results show that Cas9:guide RNA specificity extends past a 7- to 12-base pair seed sequence. Our results also suggest a tradeoff between activity and specificity both in vitro and in cells as a shorter, less-active guide RNA is more specific then a longer, more-active guide RNA. High concentrations of Cas9:guide RNA complexes can cleave off-target sites containing mutations near or within the PAM that are not cleaved when enzyme concentrations are limiting.

Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape, and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains, or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcriptional activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells.

Genome editing by Cas9, which cleaves double-stranded DNA at a sequence programmed by a short single-guide RNA (sgRNA), can result in off-target DNA modification that may be detrimental in some applications. To improve DNA cleavage specificity, we generated fusions of catalytically inactive Cas9 and FokI nuclease (fCas9). DNA cleavage by fCas9 requires association of two fCas9 monomers that simultaneously bind target sites ~15 or 25 base pairs apart. In human cells, fCas9 modified target DNA sites with >140-fold higher specificity than wild-type Cas9 and with an efficiency similar to that of paired Cas9 ‘nickases’, recently engineered variants that cleave only one DNA strand per monomer. The specificity of fCas9 was at least 4-fold higher_than that of paired nickases at loci with highly similar off-target sites. Target sites that conform to the substrate requirements of fCas9 occur on average every 34 bp in the human genome, suggesting the broad versatility of this approach for highly specific genome-wide editing.