Ruminiclostridium herbifermentans Cas12f

Characteristics

Compact type V-F nuclease (415 aa) recognizing a 5'-NCCD PAM; cleaves double-stranded DNA 21-25 bp downstream, generating staggered 5' overhangs. Wild-type editing gives indel frequencies of 1-20% across tested human loci. Cryo-EM shows a bilobed homodimer with a fully formed 20-bp R-loop in which both RuvC domains remain unresolved and highly flexible, unlike the more ordered RuvC of Al3Cas12f and OsCas12f. gRNA stem 2 forms a 90-degree kink docking into the REC/WED groove and contacting PAM-interacting residues of the non-productive monomer; deleting this region (50-60 nt) significantly reduces activity. Cleavage shows little strand bias (about 45% non-target-strand-first), and RhCas12f exits the pre-cleavage EDx intermediate more slowly than Al3Cas12f or OsCas12f.

Applications: Genome editing at 5'-NCCD PAM sites in human cells; serves as a wild-type structural and kinetic reference ortholog in comparative Cas12f mechanistic studies. Protein and sgRNA engineering (L270R paired with an optimized Rh-sg1.1 sgRNA, yielding enRhCas12f1) narrows the PAM to 5'-CCD and raises average indel frequency to 23.3%, with no detectable off-target activity by PEM-seq at the PCSK9 locus.

Limitations: Wild-type editing efficiency is modest (1-20% indels across tested human loci), well below rates achieved by engineered derivatives such as enRhCas12f1. Requires a 5'-NCCD PAM, restricting targetable sites to C-rich sequences. Both RuvC domains remain structurally unresolved with little strand-cleavage bias, so wild-type off-target behavior is not yet characterized.

References

1. Kong et al. (2023). Engineered CRISPR-OsCas12f1 and RhCas12f1 with robust activities and expanded target range for genome editing. Nat Commun - Kong 2023 Cas12f Orthologs
2. 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