RNASeq to investigate SOX2 and SOX9 function in human fetal lung tip progenitor cells. RNASeq to investigate SOX2 and SOX9 function in human fetal lung tip progenitor cells

In order to understand transcription factor SOX2 and SOX9 function, we have knocked down SOX2 and SOX9 in human fetal lung tip progenitor organoids using an inducible CRISPRi system. We discovered that SOX2 knockdown didn't appear to influence organoid cell survival and led to minimal gene expression changes at transcriptome level. By contrast, SOX9 knockdown led to organoid cell self-renewal defects and we have detected hundreds of genes differentially expressed after SOX9 knockdown. Overall design: We used 2 independent organoid parental lines infected with inducible CRISPRi system. 2 different non-targeting control (NT) gRNAs, which do not bind to anywhere of the genome, 2 different gRNAs targeting for SOX2 knockdown, and 2 different gRNAs targeting for SOX9 knockdown were used to control non-specific effect from organoid lines, lentiviral infection and different gRNAs.

为探究转录因子SOX2与SOX9的功能，我们利用诱导型CRISPR干扰（CRISPRi）系统在人类胎儿肺尖端祖器官类器官中敲低SOX2和SOX9的表达。实验发现，SOX2敲低并未对器官类器官的细胞存活产生显著影响，且在转录组水平仅引发极微弱的基因表达变化。与之形成鲜明对比的是，SOX9敲低会导致器官类器官细胞出现自我更新缺陷，我们还检测到SOX9敲低后存在数百个差异表达基因。实验总体设计：我们采用了2株独立的携带诱导型CRISPRi系统的器官类器官亲本株系。为控制器官类器官株系、慢病毒感染以及不同向导RNA（gRNA）带来的非特异性效应，本实验设置了2种不结合基因组任何区域的非靶向对照（NT）gRNA、2条靶向SOX2的敲低gRNA，以及2条靶向SOX9的敲低gRNA。