Comparative characterization of Cas12f orthologs reveals mechanistic features underlying enhanced genome editing efficiency

Guan et al. (2025). Nat. Struct. Mol. Biol. DOI: 10.1038/s41594-026-01788-6 Citations: 3

Miniature Cas12f nucleases suit AAV delivery but edit less efficiently than Cas9. This paper characterized a new ortholog, Al3Cas12f, by cryo-EM and kinetics, then engineered an RKK variant. RKK raised average SOD1 editing from under 10% to over 80%, with 26-fold gains at EMC6.

Parts used

Al3Cas12f NucleaseAl3Cas12f RKK NucleaseOsCas12f NucleaseRhCas12f Nuclease

Key findings

  • Al3Cas12f from Alistipes sp. achieved 19 of 27 guides with >50% editing in K562 cells and outperformed OsCas12f at six of eight genomic loci. The nuclease recognizes TTTV PAMs, cleaves target DNA at 20-23 nt and non-target strand at 11 nt, and exists as an obligate dimer with a naturally optimized 134-nt gRNA scaffold lacking stem 4. Active-site titration and size-exclusion chromatography confirmed stable dimerization without gRNA, with editing efficiency peaking at 19-nt spacer length across AAVS1, APOA1, and SOD1 targets.

  • Cryo-EM structures revealed Al3Cas12f extended REC helices (α2, α3) create mortise-and-tenon joints with twice the interface area of OsCas12f and RhCas12f, enabling 20-bp R-loop formation before RuvC.2 docking. OsCas12f requires RuvC.2 docking at 8-bp intermediate for full R-loop through two-lid opening (residues 229-231, 334-343). Kinetic fitting showed Al3Cas12f R-loop formation (Keq=2, 0.64 s⁻¹) with fastest cleavage rates, 65% non-target strand preference, and no partitioning into nonproductive EDx/XDP1 states that slow other orthologs.

  • Structure-guided engineering produced Al3Cas12f RKK (K79R;M190K;E222K) with >80% editing across loci at 125 ng mRNA and 50 pmol gRNA, representing one-quarter standard dosage. RKK showed up to 26-fold activity increase at EMC6 locus versus wild-type, with six of eight reference guides achieving >80% indels compared to wild-type's <61% average. The engineered variant overcame locus-dependent variability observed at SOD1 and other sites, demonstrating consistent performance that expands feasibility of low-dose AAV-compatible therapeutic applications.

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Abstract

Miniature CRISPR-Cas12f nucleases are attractive candidates for therapeutic genome editing because of their compact size and compatibility with adeno-associated virus (AAV) delivery. However, editing efficiencies in mammalian cells are lower than those of larger systems. The extensive phylogenetic diversity of Cas12f suggests unexplored mechanistic variation with the potential for optimization. Here we identify and characterize a naturally occurring Cas12f ortholog discovered through metagenomics, Alistipes sp. Cas12f (Al3Cas12f), which supports robust genome editing in human cells. Through structural, biochemical and kinetic analyses, we compare Al3Cas12f to two recently described orthologs, Oscillibacter sp. Cas12f and Ruminiclostridium herbifermentans Cas12f. These orthologs present divergent architectures and regulatory features governing protospacer-adjacent motif recognition, guide RNA (gRNA) binding, dimerization and DNA cleavage. Notably, Al3Cas12f achieves efficient R-loop formation through a stable dimer interface and a naturally optimized gRNA. Leveraging these structural insights, we generate an engineered Al3Cas12f variant (RKK) that increases editing and improves activity across several tested genomic loci. By overcoming locus-dependent variability and an apparent potency threshold, this engineered compact editor seems to expand the feasibility of low-dose, AAV-compatible therapeutic genome editing. Our results elucidate mechanistic determinants of Cas12f activity and offer a framework for engineering compact genome editors that may bear therapeutic potential.