<i>Francisella novicida</i> Cas9 interrogates genomic DNA with very high specificity and can be used for mammalian genome editing.

Abstract

Genome editing using the CRISPR/Cas9 system has been used to make precise heritable changes in the DNA of organisms. Although the widely used <i>Streptococcus pyogenes</i> 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 <i>Francisella novicida</i> 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 mismatches to the target single-guide RNA (sgRNA) are present in the sgRNA:DNA heteroduplex. FnCas9 produces staggered cleavage, higher homology-directed repair rates, and very low nonspecific genome editing compared to SpCas9. We demonstrate FnCas9-mediated correction of the sickle cell mutation in patient-derived induced pluripotent stem cells and propose that it can be used for precise therapeutic genome editing for a wide variety of genetic disorders.