Single-cell CRISPRi transcriptome screening focusing on a subset of lncRNAs regulated by hypoxia

In order to gain new insights into the molecular functions of 6 hypoxia-regulated lncRNAs in LUAD cells we performed a single-cell CRISPRi transcriptome screening based on the CROP-Seq approach. Overall design: We transduced A549 cells expressing double repressor Krab-MeCP2-dCas9 with a mini-library containing 12 validated gRNA targeting CYTOR (also known as LINC00152), LUCAT1, MALAT1, NEAT1, SNHG12 and SNHG21 as well as the two key regulators of the hypoxic response, HIF1A and HIF2/EPAS1. Two additional guides, with no effect on the genome, were used as negative controls. The transduced dCas9-Krab-MeCP2 A549 cells were equally divided in 4 samples, which were then cultured either in normoxia or in hypoxia during 3, 6 or 24 hours. Cells from each sample were labeled with a specific barcoded antibody (HTOs), pooled, and simultaneously sequenced using droplet based scRNA-seq (10X Genomics Chromium).

为深入解析肺腺癌（LUAD，Lung Adenocarcinoma）细胞中6种缺氧调控长链非编码RNA（lncRNAs，long non-coding RNAs）的分子功能，本研究基于CROP-Seq技术开展了单细胞CRISPR干扰（CRISPR interference, CRISPRi）转录组筛选实验。
整体实验设计：我们将携带双阻遏因子Krab-MeCP2-dCas9的A549细胞转导至包含12条经过验证的向导RNA（gRNA，guide RNA）的微型文库中，该文库靶向6个长链非编码RNA靶点：CYTOR（亦称LINC00152）、LUCAT1、MALAT1、NEAT1、SNHG12、SNHG21，以及缺氧应答的两个关键调控因子HIF1A与HIF2/EPAS1。此外，设置2条无基因组靶向效应的向导RNA作为阴性对照。将转导后的dCas9-Krab-MeCP2 A549细胞均分为4组样本，分别置于常氧或缺氧环境中培养3、6或24小时。随后，对每一组样本的细胞使用带有特异性条形码的抗体（HTOs）进行标记，将标记后的细胞混合后，依托基于液滴的单细胞RNA测序（scRNA-seq，single-cell RNA sequencing）技术及10X Genomics Chromium平台完成同步测序。