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AlphaFold3-guided tracrRNA redesign yields small monomeric Cas12f RNPs.
Lulu Pan1,2,3,4, Rui Sang1,2, Ruier Xue1,2
1School of Biomedical Engineering, Faculty of Engineering, University of New South Wales, Sydney 2052, Australia.
Nucleic Acids Research
|May 4, 2026
Summary
Researchers created a smaller, more active Cas12f CRISPR enzyme by modifying its guide RNA. This monomeric Cas12f enhances gene editing and biosensing applications by improving efficiency and sensitivity while maintaining a compact size.
Area of Science:
- Molecular Biology
- Biotechnology
- CRISPR Technology
Background:
- Cas12f (Cas14) is a small CRISPR effector that forms inactive dimers, limiting its use in gene editing and biosensing.
- Dimerization increases the functional size of Cas12f ribonucleoprotein (RNP) complexes, hindering applications requiring compact enzymes.
Purpose of the Study:
- To investigate the structural and functional roles of Cas12f dimerization.
- To engineer a monomeric Cas12f RNP complex for improved gene editing and biosensing capabilities.
Main Methods:
- Computational modeling and experimental validation were used to analyze Cas12f dimerization.
- A truncated tracrRNA was designed to disrupt dimer formation, creating a monomeric RNP.
Main Results:
- The truncated tracrRNA enabled the formation of a functionally monomeric Cas12f RNP.
- The monomeric RNP showed significantly enhanced trans-cleavage activity (4.5-fold for ssDNA, 3.5-fold for dsDNA, 2.5-fold for RNA).
- Detection sensitivity improved 10-fold for ssDNA/dsDNA and 4-fold for RNA, with comparable cis-cleavage and gene editing efficiency to the dimeric form.
Conclusions:
- A functionally monomeric Cas12f RNP was successfully engineered, overcoming limitations of the wild-type enzyme.
- The engineered Cas12f RNP offers enhanced biosensing performance and retains its compact size for efficient in vivo gene editing.
