Combination of engineered FnCas9 and extended gRNAs for PAM-flexible, robust and nucleobase specific editing and diagnostics

Abstract

Abstract 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 enhanced FnCas9 (enFnCas9) variants 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 increasing their accessibility across human genomic sites by ~ 3.5-fold. The enFnCas9 proteins harbor single mismatch specificity both in vitro and in cellulo leading to broadened target range of FnCas9-based CRISPR diagnostics for detection of point mutations and pathogenic DNA signatures. 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). Remarkably, enFnCas9 variants can be combined with extended length gRNAs for robust base editing at sites which are inaccessible to PAM-constrained canonical base editors. Finally, we show the complete correction of a disease-specific Retinitis Pigmentosa mutation in patient derived iPSCs using enFnCas9 Adenine Base Editor highlighting its broad application in therapeutics and diagnostics.