Structure-guided engineering of eukaryotic Fanzor-ωRNA system enhances in vivo gene editing for dystrophin restoration in a Duchenne mouse model

However, Fanzor-ωRNA system has not been well characterized in terms of gene editing activity, off-target profile and in vivo efficacy for disease correction. In the present study, Fanzor and other TnpB-ωRNA systems were reconstituted in mammalian cells for gene editing and off-target analysis.

Furthermore, we identified several Fanzor variants with enhanced activity via structure-guided protein engineering. Additionally, we demonstrated the efficacy of engineered Fanzor-ωRNA system in embryos to generate mutant mice via embryonic injection of Fanzor messenger RNA (mRNA) and ωRNA. Notably, single adeno-associated virus (AAV) delivery of engineered Fanzor-ωRNA system in a mouse model of Duchenne muscular dystrophy (DMD) achieved sufficient gene editing of DMD gene mutations to effectively induce mutant exons skipping and restore dystrophin expression, indicating the applications potential for single AAV gene editing therapy with Fanzor-ωRNA system.