Combinatorial protein engineering identifies potent CRISPR activators with reduced toxicity

Current protein engineering methods are inadequate to explore the combinatorial potential offered by nature's vast repertoire of protein domains-limiting our ability to create optimal synthetic tools. To overcome this barrier, we develop an approach to create and test thousands of chimeric proteins and employ it to probe an expansive combinatorial landscape of over 15,000 multi-domain CRISPR activators. Our findings indicate that many activators produce substantial cellular toxicity, often unrelated to their capacity to regulate gene expression. We also explore the biochemical features of activation domains and determine how their combinatorial interactions shape activator behavior. Finally, we identify two potent CRISPR activators, MHV and MMH, and demonstrate their enhanced activity across diverse targets and cell types compared to the gold-standard MCP activator, synergistic activation mediator (SAM).

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Publication Details

Journal
Nature Communications
Published
2025-11-20
DOI
https://doi.org/10.1038/s41467-025-65986-4
Primary Topic
CRISPR and Genetic Engineering
Type
article
Field-Weighted Citation Impact
0.00

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article

Combinatorial protein engineering identifies potent CRISPR activators with reduced toxicity

Antoine Forget, Yiming Huang, Harris H. Wang, Thomas Falconer et al.
Nature Communications
CRISPR and Genetic Engineering
article

Combinatorial protein engineering identifies potent CRISPR activators with reduced toxicity

Antoine Forget, Yiming Huang, Harris H. Wang, Thomas Falconer, Alejandro Chavez, Nevan J. Krogan, Mark B. De Los Santos, Gwendolyn Μ. Jang, Kam W. Leong, Cheng Qin, Yeh‐Hsing Lao, Marla J. Giddins, Max V. Staller, Tomasz Blazejewski, Alexander F. Kratz, Lai Wei, Richa Tiwari
article en

Abstract

Current protein engineering methods are inadequate to explore the combinatorial potential offered by nature's vast repertoire of protein domains-limiting our ability to create optimal synthetic tools. To overcome this barrier, we develop an approach to create and test thousands of chimeric proteins and employ it to probe an expansive combinatorial landscape of over 15,000 multi-domain CRISPR activators. Our findings indicate that many activators produce substantial cellular toxicity, often unrelated to their capacity to regulate gene expression. We also explore the biochemical features of activation domains and determine how their combinatorial interactions shape activator behavior. Finally, we identify two potent CRISPR activators, MHV and MMH, and demonstrate their enhanced activity across diverse targets and cell types compared to the gold-standard MCP activator, synergistic activation mediator (SAM).

Nature CommunicationsVol. 16(1)
Gladstone Institutes (US), University of California, San Francisco (US), University of Cambridge (GB), Columbia University Irving Medical Center (US), UC San Diego Health System (US), The Gurdon Institute (GB), University of California San Diego (US), Quantitative BioSciences (US), Chan Zuckerberg Initiative (United States) (US), University at Buffalo, State University of New York (US), Columbia University (US), University of California, Berkeley (US)
National Institutes of Health, Advanced Research Projects Agency, National Cancer Institute
Openalex Percentile: Top 68%
CRISPR and Genetic Engineering
DOIOpen Access PDFOpenAlex
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