Unravelling epigenetic drivers of chemo-resistance by optical editing of DNA-methylation in cells

DNA甲基化异常是卵巢癌获得性耐药的一个公认的驱动因素，尽管这种表观基因组变化发生在乘客事件的高背景下，并且当前的表观遗传学化学生物学工具通常影响整个表观基因组而不是特定的基因位点。这使得目前的生物学工具在研究关键基因位点特异性甲基化事件在抗性机制中的作用方面具有挑战性。为了克服这一点，使用融合到表观遗传修饰物的CRISPRCas9的表观遗传编辑正在得到越来越多的评估，尽管目前的方法具有不可诱导的局限性。为了确定抗性的关键驱动因素并展示表型效应，我们的目标是通过CRISPRCas9产生新型的化学配体，使DNA甲基化写入者和擦除者(例如DNMTS和TETS)能够通过CRISPRCas9在特定的基因组座位上进行光控定位。这些化学工具将解开特定部位甲基化在患者来源的细胞系和异种移植中的作用。表观遗传修饰物的光控制定位将使表观遗传修饰的时间控制成为可能，从而允许以时间依赖的方式研究耐药性的演变及其预防或再敏化，这对于研究获得性化疗耐药等基于表观遗传的动态过程至关重要。

Aberrant DNA-methylation is a well-established driver of acquired resistance in ovarian cancer, although such epigenomic changes occur within a high background of passenger events and current epigenetic chemical biology tools generally affect the entire epigenome rather than locus specific. This makes it challenging with current biological tools to investigate the role of key locus specific methylation events in resistance mechanisms. To overcome this, epigenetic editing using CRISPRCas9 fused to epigenetic modifiers is increasingly being evaluated, although current approaches have the limitation of not being inducible. To identify key drivers of resistance and demonstrate phenotypic effects, we aim to generate novel chemical ligands that enable light-controlled positioning of DNA methylation writer and eraser (e.g. DNMTs and TETs) at specific genomic loci by means of CRISPRCas9. These chemical tools will disentangle the role of site-specific methylation in patient-derived cell-lines and xenografts. The light-controlled positioning of the epigenetic modifiers will enable temporal control of epigenetic modification, allowing evolution of resistance and its prevention or resensitisation to be studied in a time-dependent manner that is essential to study dynamic epigenetic based processes such as acquired resistance to chemotherapy.