一种微 RNA 诱导的 CRISPR-Cas9 平台可作为微 RNA 传感器和细胞类型特异性基因组调控工具。

A microRNA-inducible CRISPR-Cas9 platform serves as a microRNA sensor and cell-type-specific genome regulation tool.

机构信息

Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking University, Beijing, China.

出版信息

Nat Cell Biol. 2019 Apr;21(4):522-530. doi: 10.1038/s41556-019-0292-7. Epub 2019 Feb 25.

DOI:10.1038/s41556-019-0292-7

PMID:30804503

Abstract

microRNAs (miRNAs) are small noncoding RNAs that play important regulatory roles in plants, animals and viruses. Measuring miRNA activity in vivo remains a big challenge. Here, using an miRNA-mediated single guide RNA (sgRNA)-releasing strategy and dCas9-VPR to drive a transgene red fluorescent protein, we create an miRNA sensor that can faithfully measure miRNA activity at cellular levels and use it to monitor differentiation status of stem cells. Furthermore, by designing sgRNAs to target endogenous loci, we adapted this system to control the expression of endogenous genes or mutate specific DNA bases upon induction by cell-type-specific miRNAs. Finally, by miRNA sensor library screening, we discover a previously undefined layer of heterogeneity associated with miR-21a activity in mouse embryonic stem cells. Together, these results highlight the utility of an miRNA-induced CRISPR-Cas9 system as miRNA sensors and cell-type-specific genome regulation tools.

摘要

microRNAs (miRNAs) 是在植物、动物和病毒中发挥重要调控作用的小非编码 RNA。在体内测量 miRNA 的活性仍然是一个巨大的挑战。在这里，我们使用 miRNA 介导的单指导 RNA（sgRNA）释放策略和 dCas9-VPR 驱动转基因红色荧光蛋白，创建了一个 miRNA 传感器，可在细胞水平上准确测量 miRNA 的活性，并将其用于监测干细胞的分化状态。此外，通过设计靶向内源性基因座的 sgRNA，我们将该系统适应于在细胞类型特异性 miRNA 的诱导下控制内源性基因的表达或突变特定的 DNA 碱基。最后，通过 miRNA 传感器文库筛选，我们发现了与 miR-21a 在小鼠胚胎干细胞中的活性相关的一个以前未定义的异质性层。总之，这些结果突出了 miRNA 诱导的 CRISPR-Cas9 系统作为 miRNA 传感器和细胞类型特异性基因组调控工具的实用性。