Liang Ziyan, Maddineni Aakaanksha, Ortega Jesus A, Magdongon Christine B, Jambardi Shreya, Roy Subrata, Tycko Josh, Patil Ajinkya, Manzano Mark, Bartom Elizabeth T, Gottwein Eva
Department of Microbiology-Immunology, Northwestern University, Feinberg School of Medicine, Chicago, IL, USA.
Department of Neurobiology, Harvard University, Boston, MA, USA.
Nat Commun. 2025 Aug 29;16(1):8071. doi: 10.1038/s41467-025-63570-4.
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.
基于CRISPR的转录激活（CRISPRa）具有广泛的研究和临床潜力。在此，我们表明常用的CRISPRa系统可表现出明显的细胞毒性。我们证明了表达转录因子p65和HSF1激活域（AD）的CRISPRa载体的毒性，p65和HSF1是协同激活介质（SAM）CRISPRa系统的组成部分。基于我们对SAM系统的研究结果，我们将研究扩展到其他AD和乙酰转移酶核心结构域。我们表明，在慢病毒生产细胞中表达强效转录激活剂会导致慢病毒滴度降低，而在转导的靶细胞中表达则会导致细胞死亡。使用诱导型慢病毒载体，我们无法确定基于SAM的有效CRISPRa的激活剂表达窗口而不产生可测量的毒性。当前基于SAM的CRISPRa系统的毒性阻碍了它们在生物医学研究中的广泛应用，并引入了可能混淆基因筛选的选择压力。我们的结果表明，CRISPRa技术的进一步发展应同时考虑基因激活效率和激活剂毒性。
