Multi-color RNA imaging with CRISPR-Cas13b systems in living cells

Liang-Zhong Yang; Bao-Qing Gao; Youkui Huang; Ying Wang; Li Yang; Ling-Ling Chen

doi:10.1016/j.cellin.2022.100044

Multi-color RNA imaging with CRISPR-Cas13b systems in living cells

Cell Insight. 2022 Jun 15;1(4):100044. doi: 10.1016/j.cellin.2022.100044. eCollection 2022 Aug.

Authors

Liang-Zhong Yang¹, Bao-Qing Gao², Youkui Huang¹, Ying Wang², Li Yang^{3

4}, Ling-Ling Chen^{1

4

5}

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, Shanghai, China.
² CAS Key Laboratory of Computational Biology, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China.
³ Center for Molecular Medicine, Children's Hospital, Fudan University and Shanghai Key Laboratory of Medical Epigenetics, International Laboratory of Medical Epigenetics and Metabolism, Ministry of Science and Technology, Institutes of Biomedical Sciences, Fudan University, Shanghai, China.
⁴ School of Life Science and Technology, ShanghaiTech University, Shanghai, China.
⁵ School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, China.

Abstract

Visualizing RNA dynamics is important for understanding RNA function. Catalytically dead (d) CRISPR-Cas13 systems have been established to image and track RNAs in living cells, but efficient dCas13 for RNA imaging is still limited. Here, we analyzed metagenomic and bacterial genomic databases to comprehensively screen Cas13 homologies for their RNA labeling capabilities in living mammalian cells. Among eight previously unreported dCas13 proteins that can be used for RNA labeling, dHgm4Cas13b and dMisCas13b displayed comparable, if not higher, efficiencies to the best-known ones when targeting endogenous MUC4 and NEAT1_2 by single guide (g) RNAs. Further examination of the labeling robustness of different dCas13 systems using the GCN4 repeats revealed that a minimum of 12 GCN4 repeats was required for dHgm4Cas13b and dMisCas13b imaging at the single RNA molecule level, while >24 GCN4 repeats were required for reported dLwaCas13a, dRfxCas13d and dPguCas13b. Importantly, by silencing pre-crRNA processing activity of dMisCas13b (ddMisCas13b) and further incorporating RNA aptamers including PP7, MS2, Pepper or BoxB to individual gRNAs, a CRISPRpalette system was developed to successfully achieve multi-color RNA visualization in living cells.

Keywords: GCN4; NEAT1; Pre-crRNA processing; RNA aptamer; RNA tracking; dCas13.