Francisella novicida Cas9 interrogates genomic DNA with very high specificity and can be used for mammalian genome editing

SUMMARY Genome editing using the CRISPR Cas9 system has been used to manipulate eukaryotic DNA and make precise heritable changes. Although the widely used Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants have been efficiently harnessed for numerous gene-editing applications across different platforms, concerns remain, regarding their putative off targeting at multiple loci across the genome. Here we report that Francisella novicida Cas9 (FnCas9) shows a very high specificity of binding to its intended targets and negligible binding to off-target loci. The specificity is determined by its minimal binding affinity with DNA when up to two mismatches to the target sgRNA are present in the sgRNA:DNA heteroduplex. In vivo , FnCas9 can be used for NHEJ mediated gene disruption and HDR mediated genomic integration. We propose that FnCas9 can be used for precise therapeutic genome editing. SIGNIFICANCE STATEMENT Therapeutic genome editing has been significantly accentuated by the ease and wide applicability of CRISPR Cas9 based gene correction. However, genomic off targeting has persisted as a major setback for its clinical applications for a large number of genetic disorders. Although high fidelity versions of Cas9 have been rationally designed, they still recognize and bind to off targets without cleaving. In this study, we characterize a naturally occurring orthogonal Cas9 from Francisella novicida (FnCas9) that shows negligible binding affinity to off targets that differ by more than two mismatches, rendering it highly specific in target recognition. Finally, we show that FnCas9 can direct both HDR and NHEJ mediated DNA repair, further expanding the toolbox for highly specific gene editing.