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Updated: Jun 18, 2026
09:16
In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
Published on: March 25, 2020
Directed evolution of compact RNA-guided nucleases for enhanced activity in mammalian cells
Fedor Gorbenko1, Irene Sala1, Young-Yoon Lee1
1Institute of Molecular Health Sciences, Department of Biology, Swiss Federal Institute of Technology (ETH) Zurich, Zurich, Switzerland.
Genome Biology
|June 17, 2026
Summary
Researchers enhanced small DNA editing enzymes, Cas12f and TnpB, using directed evolution. The resulting Cas12f1Super and TnpBSuper variants show significantly improved activity in human cells for potential gene therapy applications.
Area of Science:
- Molecular Biology
- Gene Editing Technologies
Background:
- RNA-guided nucleases, like Streptococcus pyogenes Cas9, are crucial for DNA editing but often too large for viral vector delivery.
- Smaller alternatives, such as Cas12f and TnpB families, show limited activity in mammalian cells, hindering their therapeutic potential.
Purpose of the Study:
- To improve the efficiency and utility of small RNA-guided nucleases for genome editing in mammalian systems.
- To develop enhanced variants of Cas12f and TnpB through directed evolution.
Main Methods:
- Directed evolution was employed in human cells to select for improved nuclease activity.
- Engineered variants were assessed for editing efficiency and off-target effects.
- Cas12f1Super was evaluated as a base editor compared to existing engineered variants.
Main Results:
- Directed evolution yielded Cas12f1Super and TnpBSuper variants with up to 11-fold increased editing efficiency.
- No significant increase in off-target effects was observed in the enhanced variants.
- Cas12f1Super demonstrated a tenfold improvement as a base editor compared to CasMINI.
Conclusions:
- The developed Cas12f1Super and TnpBSuper represent compact and highly efficient genome editors.
- These enhanced nucleases expand the toolkit for gene editing research and therapeutic applications in mammals.
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