脳のコレステロール代謝を調節する長鎖ノンコーディングRNAの機能解明

通过这次合作，我们首次建立了可用于CRISPR筛选的iPS细胞系。细胞系被设计成在负责钠通道的特定位点表达GFP。利用这个新建立的细胞系，我们从5000多个神经元中产生了单细胞RNA基因表达谱，并鉴定了1000个与神经元分化有关的长非编码RNA。接下来，我们针对这些与大规模CRISPR筛查兼容的长非编码RNA设计了sgRNA文库。实验设计还导致了评估筛选有效性的新方法的开发，其中包括优化细胞分析和细胞分类、免疫组织化学、细胞培养方案，其中我们成功地开发了一种方法，选择性地分离神经元群体，可以使用荧光标记抗体区分对遗传扰动的部分或全部反应。总体而言，细胞系、单细胞数据、CRISPR筛选试验的发展已经成功，并将导致发现新的神经元分化调节因子和钠通道调节因子。

Through this collaboration we first established iPS cell lines conducible for CRISPR screening. Cell lines were engineered to express GFP in specific loci responsible for sodium channel. Using this newly established cell line, we generated single cell RNA gene expression profiles from over 5000 neurons and identified 1000 long non-coding RNAs involved in neuron differentiation. We next designed sgRNA libraries targeting these long non-coding RNAs compatible for large-scale CRISPR screening. The experimental design also led to the development of novel assays to evaluate the effectiveness of the screening, which involved optimization of cytometry analysis and cell sorting, immunohistochemistry, cell culture protocols, where we have successfully developed a method to selectively isolate population of neurons that can distinguish partial to full responsiveness to genetic perturbations using fluorescently tagged antibodies. Overall, the development of cell lines, single cell data, CRISPR screening assay have been successful and will result in discovering novel regulators of neuronal differentiation and sodium channel regulation.