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

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 com...

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Autores principales: Yang, Liang-Zhong, Gao, Bao-Qing, Huang, Youkui, Wang, Ying, Yang, Li, Chen, Ling-Ling
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10120316/
https://www.ncbi.nlm.nih.gov/pubmed/37192858
http://dx.doi.org/10.1016/j.cellin.2022.100044
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author Yang, Liang-Zhong
Gao, Bao-Qing
Huang, Youkui
Wang, Ying
Yang, Li
Chen, Ling-Ling
author_facet Yang, Liang-Zhong
Gao, Bao-Qing
Huang, Youkui
Wang, Ying
Yang, Li
Chen, Ling-Ling
author_sort Yang, Liang-Zhong
collection PubMed
description 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.
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spelling pubmed-101203162023-05-15 Multi-color RNA imaging with CRISPR-Cas13b systems in living cells Yang, Liang-Zhong Gao, Bao-Qing Huang, Youkui Wang, Ying Yang, Li Chen, Ling-Ling Cell Insight Research article 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. Elsevier 2022-06-15 /pmc/articles/PMC10120316/ /pubmed/37192858 http://dx.doi.org/10.1016/j.cellin.2022.100044 Text en © 2022 The Authors https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Research article
Yang, Liang-Zhong
Gao, Bao-Qing
Huang, Youkui
Wang, Ying
Yang, Li
Chen, Ling-Ling
Multi-color RNA imaging with CRISPR-Cas13b systems in living cells
title Multi-color RNA imaging with CRISPR-Cas13b systems in living cells
title_full Multi-color RNA imaging with CRISPR-Cas13b systems in living cells
title_fullStr Multi-color RNA imaging with CRISPR-Cas13b systems in living cells
title_full_unstemmed Multi-color RNA imaging with CRISPR-Cas13b systems in living cells
title_short Multi-color RNA imaging with CRISPR-Cas13b systems in living cells
title_sort multi-color rna imaging with crispr-cas13b systems in living cells
topic Research article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10120316/
https://www.ncbi.nlm.nih.gov/pubmed/37192858
http://dx.doi.org/10.1016/j.cellin.2022.100044
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