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. 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).