Combinatorial protein engineering identifies potent CRISPR activators with reduced toxicity

Marla Giddins; Alexander F. Kratz; Mark B. De Los Santos; Antoine Forget; Richa Tiwari; Gwendolyn Jang; Tomasz Blazejewski; Chuyan Qin; Yiming Huang; Yeh Hsing Lao; Thomas Falconer; Kam W. Leong; Nevan Krogan; Max Staller; Harris Wang; Lai Wei; Alejandro Chavez

doi:10.1038/s41467-025-65986-4

Combinatorial protein engineering identifies potent CRISPR activators with reduced toxicity

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

Pharmaceutical Sciences

Columbia University
California Institute for Quantitative Biosciences
University of California at San Francisco
University of Cambridge
University of Cambridge
Gladstone Institutes
University of California at Berkeley
Chan Zuckerberg Biohub
University of California at San Diego

Research output: Contribution to journal › Article › peer-review

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).

Original language	English
Article number	11114
Journal	Nature Communications
Volume	16
Issue number	1
DOIs	https://doi.org/10.1038/s41467-025-65986-4
State	Published - Dec 2025

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Giddins, M., Kratz, A. F., De Los Santos, M. B., Forget, A., Tiwari, R., Jang, G., Blazejewski, T., Qin, C., Huang, Y., Lao, Y. H., Falconer, T., Leong, K. W., Krogan, N., Staller, M., Wang, H., Wei, L., & Chavez, A. (2025). Combinatorial protein engineering identifies potent CRISPR activators with reduced toxicity. Nature Communications, 16(1), Article 11114. https://doi.org/10.1038/s41467-025-65986-4

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title = "Combinatorial protein engineering identifies potent CRISPR activators with reduced toxicity",

abstract = "Current protein engineering methods are inadequate to explore the combinatorial potential offered by nature{\textquoteright}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).",

author = "Marla Giddins and Kratz, \{Alexander F.\} and \{De Los Santos\}, \{Mark B.\} and Antoine Forget and Richa Tiwari and Gwendolyn Jang and Tomasz Blazejewski and Chuyan Qin and Yiming Huang and Lao, \{Yeh Hsing\} and Thomas Falconer and Leong, \{Kam W.\} and Nevan Krogan and Max Staller and Harris Wang and Lai Wei and Alejandro Chavez",

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year = "2025",

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doi = "10.1038/s41467-025-65986-4",

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volume = "16",

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Giddins, M, Kratz, AF, De Los Santos, MB, Forget, A, Tiwari, R, Jang, G, Blazejewski, T, Qin, C, Huang, Y, Lao, YH, Falconer, T, Leong, KW, Krogan, N, Staller, M, Wang, H, Wei, L & Chavez, A 2025, 'Combinatorial protein engineering identifies potent CRISPR activators with reduced toxicity', Nature Communications, vol. 16, no. 1, 11114. https://doi.org/10.1038/s41467-025-65986-4

TY - JOUR

T1 - Combinatorial protein engineering identifies potent CRISPR activators with reduced toxicity

AU - Giddins, Marla

AU - Kratz, Alexander F.

AU - De Los Santos, Mark B.

AU - Forget, Antoine

AU - Tiwari, Richa

AU - Jang, Gwendolyn

AU - Blazejewski, Tomasz

AU - Qin, Chuyan

AU - Huang, Yiming

AU - Lao, Yeh Hsing

AU - Falconer, Thomas

AU - Leong, Kam W.

AU - Krogan, Nevan

AU - Staller, Max

AU - Wang, Harris

AU - Wei, Lai

AU - Chavez, Alejandro

PY - 2025/12

Y1 - 2025/12

N2 - 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).

AB - 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).

UR - https://www.scopus.com/pages/publications/105024783636

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DO - 10.1038/s41467-025-65986-4

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AN - SCOPUS:105024783636

SN - 2041-1723

VL - 16

JO - Nature Communications

JF - Nature Communications

IS - 1

M1 - 11114

ER -

Combinatorial protein engineering identifies potent CRISPR activators with reduced toxicity

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