Dynamic Imaging of RNA in Living Cells by CRISPR-Cas13 Systems

Liang-Zhong Yang; Yang Wang; Si-Qi Li; Run-Wen Yao; Peng-Fei Luan; Huang Wu; Gordon G Carmichael; Ling-Ling Chen

doi:10.1016/j.molcel.2019.10.024

Dynamic Imaging of RNA in Living Cells by CRISPR-Cas13 Systems

Mol Cell. 2019 Dec 19;76(6):981-997.e7. doi: 10.1016/j.molcel.2019.10.024. Epub 2019 Nov 19.

Authors

Liang-Zhong Yang¹, Yang Wang¹, Si-Qi Li¹, Run-Wen Yao¹, Peng-Fei Luan¹, Huang Wu¹, Gordon G Carmichael², Ling-Ling Chen³

Affiliations

¹ State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China.
² Department of Genetics and Developmental Biology, University of Connecticut Stem Cell Institute, University of Connecticut Health Center, Farmington, CT 06030-3301, USA.
³ State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China; School of Life Science and Technology, ShanghaiTech University, 100 Haike Road, Shanghai 201210, China. Electronic address: linglingchen@sibcb.ac.cn.

PMID: 31757757
DOI: 10.1016/j.molcel.2019.10.024

Abstract

Visualizing the location and dynamics of RNAs in live cells is key to understanding their function. Here, we identify two endonuclease-deficient, single-component programmable RNA-guided and RNA-targeting Cas13 RNases (dCas13s) that allow robust real-time imaging and tracking of RNAs in live cells, even when using single 20- to 27-nt-long guide RNAs. Compared to the aptamer-based MS2-MCP strategy, an optimized dCas13 system is user friendly, does not require genetic manipulation, and achieves comparable RNA-labeling efficiency. We demonstrate that the dCas13 system is capable of labeling NEAT1, SatIII, MUC4, and GCN4 RNAs and allows the study of paraspeckle-associated NEAT1 dynamics. Applying orthogonal dCas13 proteins or combining dCas13 and MS2-MCP allows dual-color imaging of RNAs in single cells. Further combination of dCas13 and dCas9 systems allows simultaneous visualization of genomic DNA and RNA transcripts in living cells.

Keywords: CRISPR-dCas13; MUC4; NEAT1; PguCas13b; PspCas13b; RNA dynamics; SatIII; nuclear stress bodies; paraspeckles.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Cas Systems / genetics
Cell Line, Tumor
Fluorescent Dyes / chemistry
Humans
Molecular Imaging / methods*
Mucin-4
Protein Engineering / methods
RNA / physiology*
RNA, Guide, CRISPR-Cas Systems / genetics
RNA, Long Noncoding
Ribonucleases / genetics
Ribonucleases / metabolism
Single Molecule Imaging / methods*
Staining and Labeling / methods

Substances

Fluorescent Dyes
MUC4 protein, human
Mucin-4
NEAT1 long non-coding RNA, human
RNA, Guide, CRISPR-Cas Systems
RNA, Long Noncoding
RNA
Ribonucleases