Structure and evolution-guided design of minimal RNA-guided nucleases
Abstract
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.
Article Details
Journal Info
Science
American Association for the Advancement of Science
Authors (20)
Petr Skopintsev
Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
Isabel Esain-Garcia
Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
Evan C. DeTurk
Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
Peter H. Yoon
Zehan Zhou
Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
Trevor Weiss
Department of Molecular, Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA, USA.
Maris Kamalu
Department of Molecular, Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA, USA.
Ajit Chamraj
Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
Kenneth J. Loi
Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
Conner J. Langeberg
Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
Ron S. Boger
Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
Hunter Nisonoff
Center for Computational Biology, University of California Berkeley, Berkeley, CA, USA.
Hannah M. Karp
Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
Lin-Xing Chen
Honglue Shi
Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
Kamakshi Vohra
Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
Jillian F. Banfield
Jamie H. D. Cate
Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
Steven E. Jacobsen
Department of Molecular, Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA, USA.
Jennifer A. Doudna