Defining Essential Enhancer for Pluripotent stem cells using Features Oriented CRISPR-Cas9 Screen (RNA-Seq 1)

Cis Regulatory Elements (CREs) regulate the expression of the genes in their genomic neighborhoods and influence cellular processes such as cell-fate maintenance and differentiation. To date, there remain major gaps in the functional characterization of CREs and the identification of its target genes in the cellular native environment. In this study, we performed a Features Oriented CRISPR Utilized Systematic (FOCUS) screen of Oct4-bound CREs using CRISPR/Cas9 to identify functional CREs important for pluripotency maintenance in mouse ES cells. RNA profiling of shLrrc31 mESCs(2 shRNAs) and shEV mESCs(2 EVs), RNA-seq was performed in biology replicates

顺式调控元件（Cis Regulatory Elements，CREs）可调控其基因组邻域内的基因表达，并参与细胞命运维持、细胞分化等细胞过程。截至目前，在细胞原生环境中对顺式调控元件进行功能表征，以及鉴定其靶基因的研究仍存在重大空白。本研究借助CRISPR/Cas9技术，针对结合Oct4的顺式调控元件开展面向特征的CRISPR系统性筛选（Features Oriented CRISPR Utilized Systematic，FOCUS），以鉴定小鼠胚胎干细胞中维持多能性所需的功能性顺式调控元件。本研究对转染shLrrc31的小鼠胚胎干细胞（采用2条短发夹RNA，shRNA）与转染空载体的小鼠胚胎干细胞（设置2个空载体对照，EV）进行转录谱分析，并以生物学重复的方式完成RNA测序。