Cytotoxicity of activator expression in CRISPR-based transcriptional activation systems

Ziyan Liang, Aakaanksha Maddineni, Jesus A. Ortega, Christine B. Magdongon, Shreya Jambardi, Subrata Roy, Josh Tycko, Ajinkya Patil, Mark Manzano, Elizabeth T. Bartom and Eva Gottwein ()
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Ziyan Liang: Northwestern University, Feinberg School of Medicine
Aakaanksha Maddineni: Northwestern University, Feinberg School of Medicine
Jesus A. Ortega: Northwestern University, Feinberg School of Medicine
Christine B. Magdongon: Northwestern University, Feinberg School of Medicine
Shreya Jambardi: Northwestern University, Feinberg School of Medicine
Subrata Roy: Northwestern University, Feinberg School of Medicine
Josh Tycko: Harvard University
Ajinkya Patil: Northwestern University, Feinberg School of Medicine
Mark Manzano: University of Arkansas for Medical Sciences
Elizabeth T. Bartom: Northwestern University, Feinberg School of Medicine
Eva Gottwein: Northwestern University, Feinberg School of Medicine

Nature Communications, 2025, vol. 16, issue 1, 1-15

Abstract: Abstract CRISPR-based transcriptional activation (CRISPRa) has extensive research and clinical potential. Here, we show that commonly used CRISPRa systems can exhibit pronounced cytotoxicity. We demonstrate the toxicity of CRISPRa vectors expressing the activation domains (ADs) of the transcription factors p65 and HSF1, components of the synergistic activation mediator (SAM) CRISPRa system. Based on our findings for the SAM system, we extended our studies to additional ADs and acetyltransferase core domains. We show that the expression of potent transcriptional activators in lentiviral producer cells can lead to low lentiviral titers, while their expression in the transduced target cells leads to cell death. Using inducible lentiviral vectors, we could not identify an activator expression window for effective SAM-based CRISPRa without measurable toxicity. The toxicity of current SAM-based CRISPRa systems hinders their wide adoption in biomedical research and introduces selection pressures that may confound genetic screens. Our results suggest that the further development of CRISPRa technology should consider both the efficiency of gene activation and activator toxicity.

Date: 2025
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DOI: 10.1038/s41467-025-63570-4

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