Oscillibacter sp. Cas12f1 (OsCas12f1)

Characteristics

Type V-F CRISPR nuclease of 433 amino acids that functions as an obligate homodimer and recognizes a 5'-YTTH PAM (Y = C or T, H = not G) in vitro. Wild-type genomic editing produces 1-20% indels across tested human loci; the crRNA-tracrRNA guide is fused via a GAAA tetraloop into a four-stem-loop scaffold with a 20-nt spacer, and deleting stem 4 (Δ106-125 nt) increases activity 10-fold. The RuvC.1 active site cleaves both DNA strands, but full 20-bp R-loop formation requires the RuvC.2 domain to first dock at an 8-bp intermediate through a two-lid opening mechanism (residues 229-231 and 334-343); cleavage kinetics are biphasic, and 70% of complexes cleave the non-target strand first.

Applications: Genomic editing demonstrated at multiple endogenous loci in human cells, with indel frequencies of 1-20% for wild-type OsCas12f1. Guide-RNA stem-loop truncations and RuvC lid mutants have been assayed in E. coli plasmid-interference and GFP-depletion assays to dissect Cas12f activation mechanisms.

Limitations: Native editing efficiency is modest and locus-variable (1-20% indels); the engineered enOsCas12f1 variant (D52R;T132R substitutions with an optimized sgRNA) is markedly more active and is the form validated for single-AAV9 in vivo delivery. Underperforms Al3Cas12f at six of eight tested genomic loci in human cells.

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