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

Background Eukaryotic Fanzor (Fz) proteins are compact RNA-guided endonucleases homologous to prokaryotic TnpB/Cas12, offering theoretical advantages for single AAV delivery. However, their low editing efficiency and poorly defined specificity in mammals have hindered therapeutic development. Results Here, we perform structure-guided engineering of Spizellomyces punctatus Fanzor1 nuclease (SpuFz1) to enhance its utility for in vivo genome editing and identify several Fanzor variants whose efficiencies are comparable or only slightly lower than widely-used miniature enAsCas12f1 and large-sized SpCas9. We demonstrate that catalytically dead dSpuFz1-v4.2 can be repurposed for CRISPRa-mediated transcriptional activation in a proof-of-concept manner. Genome-wide PEM-seq in mice reveal low translocation frequencies (below 0.05%, much lower than the background threshold set by the PEM-seq protocol), with no detected events attributable to off-target editing. GUIDE-seq analysis also confirms genome-wide low off-target profile for SpuFz1-v4.2 in human cells, with small amounts of off-target editing due to 3’ terminal sequence tolerability. Embryonic mRNA injection efficiently generates mutant mice. Crucially, single AAV9 delivery of SpuFz1-v4.2 in a humanized Duchenne muscular dystrophy model induces targeted exon skipping, restores dystrophin expression to ~ 10% of wild-type levels, and improves muscle contractile force. Conclusions Structure-guided optimization overcomes the activity barrier of eukaryotic Fanzors while maintaining low off-target effect. The engineered SpuFz1-v4.2 retains the hypercompact size necessary for single AAV packaging, establishing a robust platform for in vivo gene editing. This work positions Fanzor by a preliminary proof-of-concept therapeutic study as a viable, miniature alternative to CRISPR-Cas systems for genetic disease intervention and functional genomics.