Genome-wide CRISPR library screening to identify genes involved in the regulation of the kinetic properties of transcriptional bursting. Genome-wide CRISPR library screening to identify genes involved in the regulation of the kinetic properties of transcriptional bursting

Cell-to-cell heterogeneity in gene expression can even be observed in the same type of cells, present in a similar environment. Transcriptional bursting is thought to be one of contributing factors to the heterogeneity, but it remains elusive how the kinetic properties of transcriptional bursting (e.g. burst size, burst frequency, and noise induced by transcriptional bursting) are regulated in mammalian cells. To unbiasedly identify genes regulating the kinetic properties of transcriptional bursting, we performed large-scale CRISPR/-Cas9 based screening and functional analysis, and found that Akt/MAPK signaling pathways are involved in the regulation of the kinetic properties of transcriptional bursting. Overall design: Genome-wide CRISPR library screening was performed in Nanog, Dnmt3l, Trim28 knockin cell lines, in which GFP and iRFP reporter separately inserted into the two alleles. There are two technical replication in FACS-sorted sampels.

即便处于相似微环境中的同种细胞，也可观测到基因表达的细胞间异质性。转录爆发（transcriptional bursting）被认为是促成该异质性的因素之一，但目前尚不明确哺乳动物细胞中，转录爆发的动力学特性（例如爆发规模、爆发频率及转录爆发引发的表达噪声）是如何被调控的。为无偏地识别调控转录爆发动力学特性的基因，本研究开展了基于CRISPR-Cas9的大规模筛选与功能分析，发现Akt/MAPK信号通路参与调控转录爆发的动力学特性。实验设计：本研究在Nanog、Dnmt3l、Trim28基因敲入细胞系中开展全基因组CRISPR文库筛选，该细胞系的两个等位基因分别插入了GFP与iRFP报告基因。流式细胞分选（Fluorescence-Activated Cell Sorting, FACS）得到的样本设置了2次技术重复。