Engineered Al3Cas12f variant carrying K79R, M190K, and E222K substitutions (RKK)

Characteristics

Engineered variant of Al3Cas12f carrying three substitutions (K79R, M190K, E222K) identified through structure-guided design on Al3Cas12f's cryo-EM structure, increasing positive charge near the DNA/gRNA binding interface. Achieves >80% editing efficiency across tested genomic loci using 125 ng mRNA and 50 pmol gRNA, one-quarter the standard dose. Produces up to a 26-fold increase in editing activity at the EMC6 locus over wild-type Al3Cas12f, with six of eight reference guides reaching >80% indels versus an average below 61% for wild-type. Overcomes locus-dependent variability observed with the parental enzyme, notably at SOD1.

Applications: Low-dose genome editing where dosage-dependent toxicity or vector payload capacity is limiting, including AAV-compatible therapeutic gene editing. Preferred over wild-type Al3Cas12f when consistent high-efficiency editing is required across multiple genomic loci rather than only at favorable sites.

Limitations: Off-target activity and long-term expression stability have not been reported for the RKK variant specifically. As a 2025 engineering result, it lacks independent replication outside the originating study. Like wild-type Al3Cas12f, it requires a TTTV PAM, which constrains targetable sites relative to Cas9 orthologs with NGG or broader PAM recognition.

References

1. Guan et al. (2025). Comparative characterization of Cas12f orthologs reveals mechanistic features underlying enhanced genome editing efficiency. Nat. Struct. Mol. Biol. - Guan 2025 Cas12f Orthologs