Structure and evolution-guided design of minimal RNA-guided nucleases

• 成果类型: Article
• 署名作者: Skopintsev, Petr; Esain-Garcia, Isabel; DeTurk, Evan C.; Yoon, Peter H.; Zhou, Zehan; Weiss, Trevor; Kamalu, Maris; Chamraj, Ajit; Loi, Kenneth J.; Langeberg, Conner J.; Boger, Ron S.; Nisonoff, Hunter; Karp, Hannah M.; Chen, Lin-Xing; Shi, Honglue; Vohra, Kamakshi; Banfield, Jillian F.; Cate, Jamie H. D.; Jacobsen, Steven E.; Doudna, Jennifer A.
• 署名单位: University of California System; University of California Berkeley; University of California System; University of California Berkeley; University of California System; University of California Berkeley; University of California System; University of California Los Angeles; University of California System; University of California Berkeley; University of California System; University of California Berkeley; University of California System; University of California Berkeley; University of California System; University of California Berkeley; Monash University; United States Department of Energy (DOE); Lawrence Berkeley National Laboratory; University of California System; University of California Los Angeles; Howard Hughes Medical Institute; University of California System; University of California Berkeley; University of California System; University of California Berkeley
• 刊物名称: SCIENCE
• ISSN/ISSBN: 0036-8075; 1095-9203
• DOI: 10.1126/science.aed6123
• 发表日期: 2026-07-16
• 页码: 313-318
• 关键词: protein; bacterial; dna; crispr

摘要

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.