Al3Cas12f K79R;M190K;E222K

Al3Cas12f RKK is a structure-guided engineered variant optimized for enhanced genome editing activity. The triple mutation increases positive charge near nucleic acid binding sites, enabling consistent high-efficiency editing across diverse genomic targets.

Length: 1464 bp(488 aa)

PAM: TTTV

Cut type: 5-prime overhang

Effector domain: RuvC

Origin: Alistipes sp. (engineered)

Characteristics

Structure-guided engineered variant of Al3Cas12f with K79R, M190K, and E222K substitutions. Achieves >80% editing efficiency across tested loci at one-quarter standard dosage (125 ng mRNA, 50 pmol gRNA). Shows up to 26-fold activity increase at EMC6 locus versus wild-type. Six of eight reference guides achieve >80% indels. Enhanced positive charge at residues in hydrogen-bonding distance to DNA/gRNA substrates. Overcomes locus-dependent variability observed in wild-type.

Applications

Low-dose therapeutic genome editing, AAV-mediated gene therapy where payload space is critical. Preferred over wild-type Al3Cas12f for applications requiring consistent editing across multiple loci. Suitable for dose-sensitive therapeutic applications.

Limitations

Engineering focused on increasing charge near DNA/gRNA contact points. Long-term stability and off-target profile require validation. Slower cleavage kinetics compared to SpCas9. Relatively new variant with limited field testing.

Sequence

atggccacggagtatacatgcatcactcggaaaattgaagtccacctgcaccgtcatggtgagtatgaagaggccaagcaacgcctgattgacgactaccgggtgtgggataccattaatgataatctttacaaggccgctaatcgaattgtctcccactgctttttcaatgatgcgtatgagtacaggttaaagatccactctcctaggtttcaagaaatcgagaagctgctgcgctatccgaaacggaataagctcactgacgaggatattaaacagctgaaagccgaaaggaagcagctgttcgccgactttaaaaagcaaagacacaccttcttgagaggtggtgtcgccgaaggtgccaatcccgaacagaactcaacttacaaagtaattagcaacgaatttttagaggtcatccccagtgagatcctcaccaacctcaaccaaaacatttctagcacctataagaactattctctggacgtcgagagaggcataagaactattcccaactacaaaagaggcatccccgtgcccttctccatcaagcagaggggagagctgaagctgaagtcacgcgatgatggatccatttacgtaagattccccctgggcctcgaatgggatctctccttcggtagggaccgttcaaacaaccggaagatcgtggagagagttctttctggccaatatgacgtgggaaattcttccatccaggagtctaaaaatcgaaaacgctttctgttattggtggtgaagatacccaaagaaaaccataatctgaacccggaccggatcgtgggagtggatcttggcatcaacatccccctgtacgctgcactgaatgataacgattacgggggtatgggtattggcagccgagaacagttcctgaatatgcgcatgaggatggacgccaaaaagcgcgaactccaacggaacctgcggcagtcaacaaatggagggcacggacgcaagcagaagctccaagccctggagcgatttgaaggaaaggaaaggaactgggtgcaccttcagaatcacattttctctaagtcaatcatcgagtacgccgtgaagaataatgccggggccatacaaatggaacgcctgaccggattcgggcgtgacaagaacgacgaggttgactctaacttcaagttcattctccggtactggtctttctttgagctgcagaccatgattgagtacaaagccaacgcagccgggatcgaggtgcgctacgtggatccataccacactagccaaacatgtagcttctgcggacattatgagaaggggcagagactcaatcagagcacttttgtgtgtaaaaatcctgattgcgagaagggtaaaggtaagaaactgagtgatggaacataccaaggcatcaacgcggattggaacgcagccagaaatatcgcgctgtcagataagatagtcgaccggaaaaagaag

Literature 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

Related collections

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