Acidibacillus sulfuroxidans Cas12f1 (AsCas12f1)

Characteristics

422-amino-acid nuclease, among the smallest CRISPR-Cas effectors characterized, roughly one-third the size of SpCas9. Recognizes a 5'-NTTR PAM (R = A or G), with strongest activity at TTTG and CTTG sites, and assembles as an asymmetric homodimer bound to a single guide RNA even without target DNA present. Produces a staggered double-strand break. In Escherichia coli plasmid-interference assays, AsCas12f1 efficiently blocks transformation by low-copy plasmids, confirming native anti-plasmid CRISPR-Cas activity. In its natural, unengineered form it shows moderate genome-editing activity in human cells relative to SpCas9 and Cas12a.

Applications: Demonstrated genome editing in Escherichia coli and human cells via plasmid, ribonucleoprotein, and AAV delivery. In Bacillus anthracis, plasmid-delivered AsCas12f1 achieves complete (100%) modification of the chromosomal htrA gene and the plasmid-borne lef gene, and complete excision of a ~16.8 kb prophage, using a smaller editing plasmid than SpCas9-based approaches.

Limitations: Requires a T-rich 5'-NTTR PAM, more restrictive than SpCas9's NGG. In its native, unengineered form, human-cell editing activity is only moderate relative to SpCas9 and Cas12a. Published genome-editing applications to date are concentrated in bacteria (Bacillus anthracis, Escherichia coli); human-cell engineering data are comparatively limited.

References

1. Karvelis et al. (2020). PAM recognition by miniature CRISPR–Cas12f nucleases triggers programmable double-stranded DNA target cleavage. Nucleic Acids Res. - Karvelis 2020 Cas12f PAM
2. Wu et al. (2021). Programmed genome editing by a miniature CRISPR-Cas12f nuclease. Nat Chem Biol - Wu 2021 Cas12f
3. Wang et al. (2022). Efficient genome editing by a miniature CRISPR-AsCas12f1 nuclease in Bacillus anthracis. Front Bioeng Biotechnol - Wang 2022 AsCas12f1 B. anthracis