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Stepwise DNA-unwinding gates TnpB genome-editing activity
Zehan Zhou1, Iren Saffarian-Deemyad2, Honglue Shi3
1Innovative Genomics Institute, University of California, Berkeley, CA 94720, USA; Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
Molecular Cell
|August 5, 2026
Summary
Researchers studied the DNA unwinding mechanism of Youngiibacter multivorans TnpB (Ymu1 TnpB). Optimizing this enzyme stabilized key intermediate states, enhancing its genome editing capabilities in plants.
Area of Science:
- Molecular Biology
- Biochemistry
- Genome Engineering
Background:
- TnpB proteins are RNA-guided endonucleases and ancestors of CRISPR-Cas12, showing potential for genome engineering.
- The genome-editing efficiency of TnpBs is currently limited, with the factors controlling this activity not well understood.
Purpose of the Study:
- To investigate the DNA-unwinding mechanism of Youngiibacter multivorans TnpB (Ymu1 TnpB).
- To identify strategies for enhancing TnpB activity through protein engineering.
Main Methods:
- Biochemical assays
- Single-molecule assays
- Protein engineering of Ymu1 TnpB
Main Results:
- DNA unwinding by Ymu1 TnpB involves a stable intermediate before reaching an open state.
- The open state is formed inefficiently and is unstable without negative supercoiling.
- An engineered variant (Ymu1-WFR) stabilized these unwinding states, improving DNA cleavage and plant genome editing.
Conclusions:
- The study elucidates the physical basis for the limited activity of natural TnpBs.
- Stabilizing specific DNA unwinding states is a viable strategy to enhance TnpB-mediated genome targeting and editing efficiency.
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