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