基于 CRISPR-Cas13 的光激活 RNA N6 -甲基腺苷编辑

Photoactivatable RNA N -Methyladenosine Editing with CRISPR-Cas13.

机构信息

Department of Orthopedics, Tianjin University Tianjin Hospital, Tianjin, 300211, China.

Orthopedic Research Institute, Tianjin University Tianjin Hospital, Tianjin, 300050, China.

出版信息

Small. 2020 Jul;16(30):e1907301. doi: 10.1002/smll.201907301. Epub 2020 Jun 25.

Abstract

RNA has important and diverse biological roles, but the molecular methods to manipulate it spatiotemporally are limited. Here, an engineered photoactivatable RNA N -methyladenosine (m A) editing system with CRISPR-Cas13 is designed to direct specific m A editing. Light-inducible heterodimerizing proteins CIBN and CRY2PHR are fused to catalytically inactive PguCas13 (dCas13) and m A effectors, respectively. This system, referred to as PAMEC, enables the spatiotemporal control of m A editing in response to blue light. Further optimization of this system to create a highly efficient version, known as PAMEC , allows the manipulation of multiple genes robustly and simultaneously. When coupled with an upconversion nanoparticle film, the optogenetic operation window is extended from the visible range to tissue-penetrable near-infrared wavelengths, which offers an appealing avenue to remotely control RNA editing. These results show that PAMEC is a promising optogenetic platform for flexible and efficient targeting of RNA, with broad applicability for epitranscriptome engineering, imaging, and future therapeutic development.

摘要

RNA 具有重要且多样的生物学功能,但对其进行时空操控的分子方法却很有限。在这里,我们设计了一种基于 CRISPR-Cas13 的工程化光激活型 RNA N-甲基腺苷(m6A)编辑系统,以指导特定的 m6A 编辑。光诱导的异二聚化蛋白 CIBN 和 CRY2PHR 分别与无催化活性的 PguCas13(dCas13)和 m6A 效应子融合。该系统称为 PAMEC,能够响应蓝光实现 m6A 编辑的时空控制。对该系统进行进一步优化以创建高效版本,称为 PAMEC,可实现多个基因的稳健且同时操控。当与上转换纳米粒子膜结合使用时,光遗传学操作窗口从可见光范围扩展到组织穿透近红外波长,这为远程控制 RNA 编辑提供了一个很有吸引力的途径。这些结果表明,PAMEC 是一种有前途的光遗传学平台,可灵活高效地靶向 RNA,在表观转录组工程、成像和未来治疗开发方面具有广泛的适用性。

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