Date

Thursday 24 Apr 2025

Time

15:15 - 15:45

Location

BW032

Supervisor

Marco van Eijk

External supervisor

Dr. M.A.F.V. Gonçalves

Jury

Rene Olsthoorn

Aberrant epigenetic regulation has been observed in many diseases. CRISPR activation (CRISPRa) aims to selectively modify the epigenome by upregulation of epigenetically silenced genes. In recent years, many CRISPRa strategies have been developed improving its gene activation potency. Epigenome modifying effectors such as histone acetyl transferase (HAT) p300 and Ten-Eleven Translocation dioxygenase 1 catalytic domain (TET1-CD) are successful in improving the accessibility for transcription inducing factors binding, whereas active recruitment of transcription initiating factors have yielded very potent and selective gene activation. Additionally, mitigating immunogenic responses by humanizing CRISPRa has become a new strategy that enhances its suitability for therapeutic purposes. While promising developments have progressed this field, CRISPRa strategies have often been investigated in specific settings, usually comparing earlier effector versions with new versions. In this study, we took a broad multiplexing approach where we applied combinations of CRISPRa strategies in distinct genetic settings to highlight communalities and discrepancies regarding the context dependency of CRISPRa tools. We discovered that combinations of transactivation domain (TAD) recruitment with TET1-CD or p300 HAT yield synergistic gene activating potency, with the latter combination being the most potent in activating Receptor activator of nuclear factor kappa-Β ligand (RANKL). However, hitherto, the gene activating effects are not lasting and will mostly return to endogenous levels after a few days. Furthermore, placement of the effectors with respect to the transcription start site (TSS) was observed to exert significant influence over the gene activating potency. We have shown that certain genes have strong biases for effectors of human origin, evidently provided by diminished gene activation when targeted with a virus derived TAD multimer of VP64, p65, and Rta (VPR). Lastly, we highlighted effector biases in specific epigenetic settings and provided evidence of the context dependency of CRISPRa by showing that potent effector combinations can be completely inactive when targeting other genes.