Alistipes sp. Cas12f

Al3Cas12f is a naturally optimized compact CRISPR nuclease discovered from metagenomic analysis of Alistipes sp. microbiomes. It represents a highly active member of the Cas12f family with structural features enabling efficient genome editing.

Length: 1464 bp(488 aa)

PAM: TTTV

Cut type: 5-prime overhang

Effector domain: RuvC

Origin: Alistipes sp.

Characteristics

Compact Cas12f nuclease (488 aa) discovered through metagenomics. Exists as obligate dimer with naturally optimized 134-nt gRNA scaffold lacking stem 4. Extended REC domain helices (α2, α3) form mortise-and-tenon dimer interface with twice the contact area of other Cas12f orthologs. Achieves 20-bp R-loop formation before RuvC.2 docking. Optimal editing with 19-nt spacer length. Shows 65% non-target strand cleavage preference. High editing efficiency with 19 of 27 guides achieving >50% indels in K562 cells.

Applications

AAV-compatible genome editing, therapeutic applications requiring compact nucleases, single-AAV delivery systems. Suitable for mammalian cell editing across diverse genomic loci including AAVS1, APOA1, and SOD1.

Limitations

Locus-dependent editing variability observed at some targets (e.g., SOD1 <61%). May require engineering for consistent performance across all genomic contexts. Slower cleavage kinetics compared to SpCas9.

Sequence

atggccacggagtatacatgcatcactcggaaaattgaagtccacctgcaccgtcatggtgagtatgaagaggccaagcaacgcctgattgacgactaccgggtgtgggataccattaatgataatctttacaaggccgctaatcgaattgtctcccactgctttttcaatgatgcgtatgagtacaggttaaagatccactctcctaggtttcaagaaatcgagaagctgctgaaatatccgaaacggaataagctcactgacgaggatattaaacagctgaaagccgaaaggaagcagctgttcgccgactttaaaaagcaaagacacaccttcttgagaggtggtgtcgccgaaggtgccaatcccgaacagaactcaacttacaaagtaattagcaacgaatttttagaggtcatccccagtgagatcctcaccaacctcaaccaaaacatttctagcacctataagaactattctctggacgtcgagagaggcataagaactattcccaactacaaaagaggcatccccgtgcccttctccatcaagcagaggggagagctgatgctgaagtcacgcgatgatggatccatttacgtaagattccccctgggcctcgaatgggatctctccttcggtagggaccgttcaaacaaccgggagatcgtggagagagttctttctggccaatatgacgtgggaaattcttccatccaggagtctaaaaatcgaaaacgctttctgttattggtggtgaagatacccaaagaaaaccataatctgaacccggaccggatcgtgggagtggatcttggcatcaacatccccctgtacgctgcactgaatgataacgattacgggggtatgggtattggcagccgagaacagttcctgaatatgcgcatgaggatggacgccaaaaagcgcgaactccaacggaacctgcggcagtcaacaaatggagggcacggacgcaagcagaagctccaagccctggagcgatttgaaggaaaggaaaggaactgggtgcaccttcagaatcacattttctctaagtcaatcatcgagtacgccgtgaagaataatgccggggccatacaaatggaacgcctgaccggattcgggcgtgacaagaacgacgaggttgactctaacttcaagttcattctccggtactggtctttctttgagctgcagaccatgattgagtacaaagccaacgcagccgggatcgaggtgcgctacgtggatccataccacactagccaaacatgtagcttctgcggacattatgagaaggggcagagactcaatcagagcacttttgtgtgtaaaaatcctgattgcgagaagggtaaaggtaagaaactgagtgatggaacataccaaggcatcaacgcggattggaacgcagccagaaatatcgcgctgtcagataagatagtcgaccggaaaaagaag

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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