Cytotoxicity of activator expression in CRISPR-based transcriptional activation systems

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. CRISPR-based transcriptional activation (CRISPRa) is a powerful tool for controlling gene expression. Here, Liang et al. show that commonly used CRISPRa systems are surprisingly toxic to cells due to their potent activation domains, informing the application and development of this technology.