美国加州大学Jennifer A. Doudna团队的一项最新研究开发出了利用CRISPR-Csm进行单分子活细胞RNA成像。相关论文于2025年2月18日发表在《自然—生物技术》杂志上。

小组提出了单分子活细胞荧光原位杂交（smLiveFISH），这是一种将可编程RNA引导、RNA靶向的CRISPR-Csm复合体与多路引导RNA相结合的方法，可直接有效地可视化包括原代细胞在内的一系列细胞类型中的单个RNA分子。使用smLiveFISH，研究小组在活细胞中追踪单个原生NOTCH2和MAP1B转录本，并确定了两种不同的定位机制，包括NOTCH2 mRNA在内质网的共翻译易位和MAP1B mRNA向细胞外周的定向转运。这种方法有可能解开控制健康和疾病中天然转录本时空组织的原理。

据介绍，了解单个细胞中单个RNA分子的各种动态行为需要以高分辨率实时可视化。然而，未修饰的内源性RNA的单分子活细胞成像尚未以可推广的方式实现。

附：英文原文

Title: Single-molecule live-cell RNA imaging with CRISPR–Csm

Author: Xia, Chenglong, Colognori, David, Jiang, Xueyang Stephen, Xu, Ke, Doudna, Jennifer A.

Issue&Volume: 2025-02-18

Abstract: Understanding the diverse dynamic behaviors of individual RNA molecules in single cells requires visualizing them at high resolution in real time. However, single-molecule live-cell imaging of unmodified endogenous RNA has not yet been achieved in a generalizable manner. Here, we present single-molecule live-cell fluorescence in situ hybridization (smLiveFISH), a robust approach that combines the programmable RNA-guided, RNA-targeting CRISPR–Csm complex with multiplexed guide RNAs for direct and efficient visualization of single RNA molecules in a range of cell types, including primary cells. Using smLiveFISH, we track individual native NOTCH2 and MAP1B transcripts in living cells and identify two distinct localization mechanisms including the cotranslational translocation of NOTCH2 mRNA at the endoplasmic reticulum and directional transport of MAP1B mRNA toward the cell periphery. This method has the potential to unlock principles governing the spatiotemporal organization of native transcripts in health and disease.

DOI: 10.1038/s41587-024-02540-5

Source: https://www.nature.com/articles/s41587-024-02540-5