CRISPR 介导的胚胎干细胞中转座元件的激活。

CRISPR-Mediated Activation of Transposable Elements in Embryonic Stem Cells.

机构信息

Department of Molecular Biology, Keio University School of Medicine, Tokyo, Japan.

Department of Microbiology and Molecular Genetics, University of California, Davis, Davis, CA, USA.

出版信息

Methods Mol Biol. 2022;2509:171-194. doi: 10.1007/978-1-0716-2380-0_11.

DOI:10.1007/978-1-0716-2380-0_11

PMID:35796964

Abstract

Mounting evidence has established that subsets of transposable elements (TEs) function as gene regulatory elements in a cell type- and species-specific manner. Here we describe an in vitro system to ectopically activate TEs using CRISPR-mediated activation (CRISPRa) for functional studies in mouse embryonic stem cells (ESCs). We established a stable mouse CRISPRa ESC line, in which expression of guide RNA enables the activation of TE-derived enhancers and the expression of their adjacent genes. We show an example of ectopic activation of TE-derived enhancers that function in male meiosis, as well as the expression of adjacent germline genes in ESCs. This system can also be applied to functional studies of TEs that are not active in ESCs.

摘要

越来越多的证据表明，转座元件 (TEs) 的亚群以细胞类型和物种特异性的方式作为基因调控元件发挥作用。在这里，我们描述了一种使用 CRISPR 介导的激活 (CRISPRa) 在体外异位激活 TEs 的系统，用于在小鼠胚胎干细胞 (ESCs) 中进行功能研究。我们建立了一个稳定的小鼠 CRISPRa ESC 系，其中向导 RNA 的表达可激活 TE 衍生的增强子，并表达其相邻基因。我们展示了一个异位激活 TE 衍生增强子的例子，该增强子在雄性减数分裂中发挥作用，以及 ESCs 中相邻生殖系基因的表达。该系统也可应用于在 ESCs 中不活跃的 TEs 的功能研究。