Alistipes sp. Cas12f

Al3Cas12f is a compact, naturally occurring CRISPR-Cas12f nuclease from Alistipes sp. identified through metagenomic screening. It forms an obligate homodimer with a 134-nucleotide guide RNA and cleaves double-stranded DNA at TTTV PAM sites, achieving higher editing efficiency in human cells than related Cas12f orthologs.

Length: 1464 bp (488 aa)

PAM: TTTV

Cut type: 5_overhang

Effector domain: RuvC

Origin: Alistipes sp.

Characteristics

488-amino-acid nuclease that functions as an obligate homodimer even without bound gRNA, using a naturally optimized 134-nt gRNA scaffold that lacks stem 4. Extended REC domain helices (α2, α3) form a mortise-and-tenon dimer interface with twice the contact area of OsCas12f and RhCas12f, enabling a fully formed 20-bp R-loop before RuvC.2 docks, unlike OsCas12f which requires RuvC.2 docking at an 8-bp intermediate. Cleaves the non-target strand with 65% preference, shows the fastest cleavage rate among characterized Cas12f orthologs though still slower than SpCas9, and produces >50% indels for 19 of 27 guides tested in K562 cells at an optimal 19-nt spacer length.

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

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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

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