Structure and evolution-guided design of minimal RNA-guided nucleases
The design of RNA-guided nucleases with properties not limited by evolution can expand programmable genome-editing capabilities. However, generating diverse multidomain proteins with robust enzymatic properties remains challenging. Here, we use a protein design strategy that couples a structure-guided inverse-folding model with evolution-informed residue constraints to generate active, divergent variants of TnpB, a minimal CRISPR-Cas12-like nuclease, termed SynTnpBs. High-throughput screening of artificial intelligence-generated variants yielded editors that retained or exceeded wild-type activity in bacterial, plant, and human cells. Cryo-electron microscopy-based structure determination of the most divergent variant revealed stabilizing contacts in the RNA-DNA interfaces across conformations, demonstrating the design potential of this approach. Together, these results establish a strategy for creating non-natural RNA-guided nucleases and conformationally active nucleic acid binders, enlarging the designable protein space.
Authors
- Trevor Weiss (ORCID: https://orcid.org/0000-0002-2373-0802)
- Steven E. Jacobsen (ORCID: https://orcid.org/0000-0001-9483-138X)
- Lin-Xing Chen (ORCID: https://orcid.org/0000-0003-2774-1952)
- J.H.D. Cate (ORCID: https://orcid.org/0000-0001-5965-7902)
- Jillian F. Banfield (ORCID: https://orcid.org/0000-0001-8203-8771)
- Conner J. Langeberg (ORCID: https://orcid.org/0000-0002-5609-3758)
- Honglue Shi (ORCID: https://orcid.org/0000-0003-3847-1652)
- Jennifer A. Doudna (ORCID: https://orcid.org/0000-0001-9161-999X)
- Hunter Nisonoff (ORCID: https://orcid.org/0000-0003-1357-8111)
- Petr Skopintsev (ORCID: https://orcid.org/0000-0002-6043-157X)
- Hannah Karp (ORCID: https://orcid.org/0000-0002-1619-5901)
- Isabel Esain-Garcia (ORCID: https://orcid.org/0000-0003-1931-4563)
- Peter H. Yoon (ORCID: https://orcid.org/0000-0002-9156-1393)
- Kenneth J. Loi (ORCID: https://orcid.org/0009-0009-5508-6511)
- Evan C. DeTurk (ORCID: https://orcid.org/0009-0004-9613-8625)
- Ron Boger (ORCID: https://orcid.org/0000-0002-4467-271X)
- Z. S. Zhou (ORCID: https://orcid.org/0000-0003-1174-2635)
- Kamakshi Vohra (ORCID: https://orcid.org/0009-0004-4368-2694)
- Maris Kamalu
- Ajit Chamraj (ORCID: https://orcid.org/0009-0005-7114-1753)
Institutions
- QB3 (US)
- Planetary Science Institute (US)
- Gladstone Institutes (US)
- Howard Hughes Medical Institute (US)
- Lawrence Berkeley National Laboratory (US)
- University of California, Los Angeles (US)
- Australian Regenerative Medicine Institute (AU)
- Innovative Genomics Institute (US)
- Monash University (AU)
- University of California, Berkeley (US)
Publication Details
- Journal
- Science
- Published
- 2026-07-16
- DOI
- https://doi.org/10.1126/science.aed6123
- Primary Topic
- Advanced biosensing and bioanalysis techniques
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- National Science Foundation
- Howard Hughes Medical Institute
- Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung