CRISPRi DSP-kd in iAT2

We performed single cell transcriptomic profiling of induced human pluripotent stem cells (iPSCs)-derived type 2 alveolar epithelial cells (iAT2). iPSCs stably expressed CRISPRi (dCas9-KRAB) under the control of doxycyline. iAT2s were transduced with a lentivirus expressing gRNA targeting the transcriptional start site of DSP. Cells were treated with or without doxycyline to intiate CRISPRi-knockdown. Prior to capture, cells were labelled with hashing antibodies (HTO). Cells were captured for 10x Genomics Single cell capture (V3 protocol), and GEX and HTO libraries were sequenced. HTODemux function was used to demultiplex the samples. Knockdown of DSP caused cells to cluster separately from wild-type cells. Single-cell transcriptomic analysis of iPSC-derived type 2 alveolar epithelial cells (iAT2) comparing wild-type cells with CRISPRi-mediated knockdown of DSP

本研究针对诱导多能干细胞（iPSCs）衍生的2型肺泡上皮细胞（iAT2）开展单细胞转录组谱分析，对比野生型细胞与CRISPRi介导的DSP敲低细胞。实验所用的iPSCs稳定表达受多西环素调控的CRISPR干扰（CRISPRi，dCas9-KRAB）系统。将携带靶向DSP转录起始位点的向导RNA（gRNA）的慢病毒转导至iAT2细胞，随后通过添加或不添加多西环素启动CRISPRi介导的基因敲低。细胞捕获前，采用哈希抗体（HTO）完成细胞标记。采用10x Genomics单细胞捕获（V3实验方案）完成细胞捕获，并对基因表达（GEX）文库与HTO文库进行测序。使用HTODemux工具完成样本的解多路拆分。研究结果显示，DSP敲低可使细胞与野生型细胞形成独立的细胞聚类簇。