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

2020年诺贝尔化学奖授予Charpentier和Doudna，以表彰他们开发CRISPR-Cas9，这是生物技术、基因治疗和基因组编辑领域的一场革命。然而，该系统的应用在很大程度上受到效率低下的限制，特别是对原代细胞和诱导的多能干细胞，以及无意编辑造成的脱靶效应。由于其良好的药代动力学和药效学，一些小分子已被证明提高CRISPR-Cas9系统的活性和特异性。它们大多以间接方式影响CRISPR-Cas9，导致某些细胞类型的活性和效率较低。为了寻找能够直接与Cas9相互作用从而影响Cas9功能和活性的小分子，我们建立了一种基于荧光的高含量化学筛选方法。我们从一个由数百种类药物化合物组成的化学库中确定丙戊酸(VPA)是Cas9的一种不稳定因素。我们证明了VPA通过在体外直接相互作用降低了Cas9的稳定性，从而在高温条件下抑制了细胞中CRISPR-Cas9的基因组编辑，这种作用独立于它对组蛋白脱乙酰基酶的抑制作用。结合用近红外激光照射光热剂吲哚青绿或用外部热袋加热，我们实现了瞬时、可控的热疗条件，选择性地在所需位置或细胞群降解和抑制Cas9蛋白。在这个拟议的项目中，我们将继续研究Cas9-VPA复合体的结合模型。此外，从我们的筛选中，我们有趣地发现，根据它们的化学结构，在四个簇中有32个匹配，在体外显示出很强的结合亲和力。我们打算在体外、细胞和果蝇体内专门测试簇1和簇2中的化合物的活性和功能。Cas9-药物络合物的共晶结构和化学优化将独立、互补地进行。综上所述，我们不仅将找到被批准的药物作为新的Cas9调节剂，而且还将全面了解药物与Cas9的结合情况，促进CRISPR-Cas9系统的应用。

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.