Comparative characterization of Cas12f orthologs reveals mechanistic features underlying enhanced genome editing efficiency - PubMed
4 hours ago
- #Genome Editing
- #CRISPR-Cas12f
- #Therapeutic Applications
- Miniature CRISPR-Cas12f nucleases are compact and AAV-compatible, making them attractive for therapeutic genome editing, though their efficiency in mammalian cells is often low.
- A new Cas12f ortholog, Al3Cas12f from Alistipes sp., was discovered via metagenomics and shows robust genome editing in human cells.
- Comparative analysis with other Cas12f orthologs (Oscillibacter sp. and Ruminiclostridium herbifermentans) reveals divergent structural and regulatory features affecting PAM recognition, gRNA binding, dimerization, and DNA cleavage.
- Al3Cas12f achieves efficient R-loop formation due to a stable dimer interface and an optimized gRNA, leading to an engineered variant (RKK) that enhances editing efficiency across multiple genomic loci.
- This engineering advances compact editors for low-dose, AAV-compatible therapeutic applications by overcoming locus-dependent variability and potency thresholds.