Seeking for small molecules to improve the efficiency and specificity of CRISPR-Cas9-mediated genome editing

点击翻译按钮获取中文摘要

The 2020 Nobel Prize in chemistry was awarded to Charpentier and Doudna for the development of CRISPR-Cas9, a revolution in the field of biotechnology, gene therapy, and genome editing. However, the application of this system is largely limited by low efficiency, particularly for primary cells and induced pluripotent stem cells, and off-target effect caused by unintentional editing. Due to its excellent pharmacokinetics and pharmacodynamics, a number of small molecules have been shown to improve the activity and specificity of CRISPR-Cas9 system. Most of them affect CRISPR-Cas9 in an indirect manner, resulting in less activity and efficiency in certain cell types. Seeking for small molecules that can directly interact with and, thereby, affect Cas9 function and activity, we established a fluorescence-based approach to high-content chemical screening. We identified valproic acid (VPA) as a Cas9 destabilizer from a chemical library consisting of hundreds of drug-like compounds. We demonstrated that VPA reduced Cas9 stability by directly interacting in vitro and, thereby, inhibiting CRISPR-Cas9 genome editing in cells under conditions of hyperthermia, an effect that is independent from its inhibitory effect against histone deacetylase. In combination with irradiating the photothermal agent indocyanine green by a near-infrared laser or heating with an external heat bag, we achieved transient, controllable hyperthermia conditions to selectively degrade and inhibit Cas9 protein at desired places or cell population. In this proposed project, we will continue to investigate the binding model of the Cas9-VPA complex. Moreover, from our screening we interestingly found 32 hits in four clusters according to their chemical structures, showing robust binding affinity in vitro. We intend to exclusively test activities and functions of compounds in cluster 1 and 2 in vitro, in cells and in Drosophila. Co-crystal structures of Cas9-drugs complexes and chemical optimization will be independently, complementarily performed. Taken together, we will not only find approved drugs as new Cas9 modulators but also achieve a comprehensive understanding of the drug-Cas9 binding profile, facilitating the application of CRISPR-Cas9 system.