PhD defence S. (Siwei) Chen

Modulating the activity of CRISPR/Cas9 genome editing by small molecules

The use of the bacterial-derived CRISPR/Cas9 genome editing system offers enormous opportunities to treat human genetic diseases. However, the potency of CRISPR/Cas9 is limited by the low precise genome editing efficiency and continuous activity of Cas9. The development of cell permeable small molecules is promising approach for precise control of Cas9 activity and may greatly enhance the application of genome editing in academic, industrial, and clinical settings. In this thesis, we found that the efficiency of CRISPR/Cas9-mediated genome editing can be modulated by histone deacetylases (HDACs) inhibitors. In order to identify potential Cas9 inhibitors and enhancers we developed a living cell-based high-throughput screening platform. Using this assay, we identified two novel potent small molecule Cas9 inhibitors that switch off Cas9 activity as well as a new potent Cas9 enhancer. These molecules inhibited/enhanced the CRISPR/Cas9-mediated genome editing in biochemical, eukaryotic, and prokaryotic cell studies. Interestingly, the potent Cas9 enhancer was obtained by a minor variation from one of the Cas9 inhibitors. Next, we showed that the Cas9 inhibitors and enhancer can modify the Cas9 activity by binding at the RuvC active site of the Cas9 endonuclease. Our results provide a practical and possibly clinically applicable way to precisely modulate CRISPR/Cas9-mediated genome editing activity using small molecules. These findings also gain new insight into the interactions between small molecules and the Cas9 protein and may extend the application of genome editing techniques.

Promotores Prof.dr. H.J. Haisma and Prof.dr. F.J. Dekker