The clinical success of CRISPR therapies is dependent on the safety and efficacy of
Cas proteins. The Cas9 from Francisella novicida (FnCas9) has negligible affinity for
mismatched substrates enabling it to discriminate off-targets in DNA with very high
precision even at the level of binding. However, its cellular targeting efficiency is low,
limiting its use in therapeutic applications. Here, we rationally engineer the protein to
develop engineered(enFnCas9) variants with enhanced activity and expand its cellular
editing activity to genomic loci previously inaccessible. Notably, some of the variants
release the protospacer adjacent motif (PAM) constraint from NGG to NGR/NRG
making them rank just below SpCas9-RY and SpCas9-NG in their accessibility across
human genomic sites. The enFnCas9 proteins, similar to Cas12a and Cas12f, harbor
high intrinsic specificity and can diagnose single nucleotide variants accurately.
Importantly, they provide superior outcomes in terms of editing efficiency, knock-in
rates, and off-target specificity over other engineered high-fidelity versions of SpCas9
(SpCas9-HF1 and eSpCas9). Broad targeting range coupled with remarkable
specificity of DNA interrogation underscores the utility of these variants for safe and
efficient therapeutic gene correction across multiple cell lines and target loci.