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Stepwise DNA unwinding gates TnpB genome-editing activity
Zehan Zhou1,2, Iren Saffarian-Deemyad3, Honglue Shi1,4,5
1Innovative Genomics Institute, University of California, Berkeley, CA, USA, 94720.
Biorxiv : the Preprint Server for Biology
|January 16, 2026
Summary
Researchers improved genome engineering by stabilizing DNA unwinding states in TnpB enzymes. This enhances DNA cleavage and editing efficiency, overcoming limitations of natural TnpB (transposase B) systems.
Area of Science:
- Molecular Biology
- Biochemistry
- Genomics
Background:
- TnpB (transposase B) is an RNA-guided endonuclease and a precursor to CRISPR-Cas12, holding potential for genome engineering.
- The genome-editing capabilities of TnpBs are currently limited, with the factors influencing their 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 for improved genome engineering applications.
Main Methods:
- Biochemical assays were employed to analyze the DNA-unwinding process.
- Single-molecule assays were utilized to observe the dynamic states of DNA unwinding by Ymu1 TnpB.
Main Results:
- DNA unwinding by Ymu1 TnpB involves intermediate and fully unwound states, with the latter being unstable without negative supercoiling.
- An optimized variant, Ymu1-WFR, was developed that stabilizes these unwinding states.
- Ymu1-WFR demonstrated enhanced DNA cleavage in vitro and increased genome editing efficiency in vivo.
Conclusions:
- The study elucidates the physical basis for the limited activity of natural TnpBs.
- Stabilizing specific DNA unwinding states is key to enhancing TnpB-mediated DNA targeting and genome editing efficacy.
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