Stepwise DNA unwinding gates TnpB genome-editing activity
TnpB is a compact RNA-guided endonuclease and evolutionary ancestor of CRISPR-Cas12 that offers a promising platform for genome engineering. However, the genome-editing activity of TnpBs remains limited and its underlying determinants are poorly understood. Here, we used biochemical and single-molecule assays to examine the DNA-unwinding mechanism of <i>Youngiibacter multivorans</i> TnpB (Ymu1 TnpB). DNA unwinding proceeds through formation of a partially unwound intermediate state to a fully unwound open state. The open state forms inefficiently and collapses readily in the absence of negative supercoiling. An optimized variant, Ymu1-WFR, stabilizes formation of both the intermediate and open states, resulting in enhanced DNA cleavage <i>in vitro</i> and increased genome editing <i>in vivo</i>. These findings identify the physical basis for the observed minimal activities of natural TnpBs, revealing how stabilizing specific unwinding states enables efficient DNA targeting.
Authors
- Trevor Weiss (ORCID: https://orcid.org/0000-0002-2373-0802)
- David F. Savage (ORCID: https://orcid.org/0000-0003-0042-2257)
- Brittney W. Thornton (ORCID: https://orcid.org/0009-0000-5615-2853)
- Zev Bryant (ORCID: https://orcid.org/0000-0001-6751-3693)
- Rachel F. Weissman (ORCID: https://orcid.org/0000-0002-1806-6564)
- Kevin Wasko (ORCID: https://orcid.org/0000-0001-6629-6850)
- Marena Trinidad (ORCID: https://orcid.org/0000-0001-7839-4642)
- Jasmine Amerasekera
- Conner J. Langeberg (ORCID: https://orcid.org/0000-0002-5609-3758)
- Noor Al-Sayyad
- Honglue Shi (ORCID: https://orcid.org/0000-0003-3847-1652)
- Petr Skopintsev (ORCID: https://orcid.org/0000-0002-6043-157X)
- Isabel Esain-Garcia (ORCID: https://orcid.org/0000-0003-1931-4563)
- Evan C. DeTurk (ORCID: https://orcid.org/0009-0004-9613-8625)
- Kamakshi Vohra (ORCID: https://orcid.org/0009-0004-4368-2694)
- Maris Kamalu
- Iren Saffarian-Deemyad
- Zehan Zhou
- Kevin D.P. Aris
- David F. Savage
- Muhammad Moez ur-Rehman
- Steven E. Jacobsen
- Peter H. Yoon
- Erin E. Doherty
Institutions
- QB3 (US)
- Howard Hughes Medical Institute (US)
- University of California, Los Angeles (US)
- Innovative Genomics Institute (US)
- Stanford Medicine (US)
- University of California, Berkeley (US)
- Stanford University (US)
Publication Details
- Journal
- bioRxiv (Cold Spring Harbor Laboratory)
- Published
- 2026-01-09
- DOI
- https://doi.org/10.64898/2026.01.09.698545
- Citations
- 2
- Primary Topic
- CRISPR and Genetic Engineering
- Type
- article
- Field-Weighted Citation Impact
- 44.47