biostudies-literatureUnknownMaddineni ANCI NIH HHSNIH HHSCRISPR-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 published and new 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 the p300 acetyltransferase core domain. We show that the expression of potent transcriptional activators in lentiviral producer cells leads 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 bottlenecks 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.bioRxiv : the preprint server for biology2024.09.23.614524https://www.ebi.ac.uk/biostudies/studies/S-EPMC11463599biostudies-literatureCytotoxicity of Activator Expression in CRISPR-based Transcriptional Activation Systems.PMC11463599S10 OD011996S10 OD026814R01 CA247619Manzano MJambardi SLiang ZTycko JGottwein ERoy SPatil AMaddineni AfalseCytotoxicity 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 published and new 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 the p300 acetyltransferase core domain. We show that the expression of potent transcriptional activators in lentiviral producer cells leads 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 bottlenecks 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.2024-01-01T00:00:00Z2024 Sep2026-04-21T03:20:40.915Z2025-04-06T14:23:01.615ZS-EPMC114635993938651810.1101/2024.09.23.614524