利用面向特征的 CRISPR-Cas9 筛选技术定义多能干细胞的必需增强子。

Defining Essential Enhancers for Pluripotent Stem Cells Using a Features-Oriented CRISPR-Cas9 Screen.

机构信息

Laboratory for Epigenetics, Stem Cells and Cell Therapy, Programme in Stem Cell, Regenerative Medicine and Aging, A(∗)STAR Institute of Molecular and Cell Biology, Singapore 138673, Singapore; Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore.

Laboratory for Epigenetics, Stem Cells and Cell Therapy, Programme in Stem Cell, Regenerative Medicine and Aging, A(∗)STAR Institute of Molecular and Cell Biology, Singapore 138673, Singapore.

出版信息

Cell Rep. 2020 Oct 27;33(4):108309. doi: 10.1016/j.celrep.2020.108309.

DOI:10.1016/j.celrep.2020.108309

PMID:33113365

Abstract

cis-regulatory elements (CREs) regulate the expression of 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 their target genes in the cellular native environment. In this study, we perform a features-oriented CRISPR-utilized systematic (FOCUS) screen of OCT4-bound CREs using CRISPR-Cas9 to identify functional enhancers important for pluripotency maintenance in mESCs. From the initial 235 candidates tested, 16 CREs are identified to be essential stem cell enhancers. Using RNA-seq and genomic 4C-seq, we further uncover a complex network of candidate CREs and their downstream target genes, which supports the growth and self-renewal of mESCs. Notably, an essential enhancer, CRE111, and its target, Lrrc31, form the important switch to modulate the LIF-JAK1-STAT3 signaling pathway.

摘要

顺式调控元件（CREs）调节其基因组邻近区域基因的表达，并影响细胞过程，如细胞命运维持和分化。迄今为止，在细胞天然环境中，CREs 的功能特征分析和其靶基因的鉴定仍存在很大差距。在这项研究中，我们使用 CRISPR-Cas9 对 OCT4 结合的 CREs 进行了面向特征的 CRISPR 利用系统（FOCUS）筛选，以鉴定在 mESCs 中维持多能性所必需的功能增强子。在最初测试的 235 个候选者中，有 16 个 CRE 被鉴定为必需的干细胞增强子。通过 RNA-seq 和基因组 4C-seq，我们进一步揭示了候选 CREs 及其下游靶基因的复杂网络，这些基因支持 mESCs 的生长和自我更新。值得注意的是，一个必需的增强子 CRE111 和它的靶基因 Lrrc31 形成了一个重要的开关，来调节 LIF-JAK1-STAT3 信号通路。