Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs

Short hairpin RNAs (shRNAs) are used to deplete circRNAs by targeting back-splicing junction (BSJ) sites. However, frequent discrepancies exist between shRNA-mediated circRNA knockdown and the corresponding biological effect, querying their robustness. By leveraging CRISPR/Cas13d tool and optimizing...

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Autores principales: Zhang, Yang, Nguyen, Tuan M., Zhang, Xiao-Ou, Wang, Limei, Phan, Tin, Clohessy, John G., Pandolfi, Pier Paolo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2021
Materias:
Method
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7818937/
https://www.ncbi.nlm.nih.gov/pubmed/33478577
http://dx.doi.org/10.1186/s13059-021-02263-9
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author Zhang, Yang
Nguyen, Tuan M.
Zhang, Xiao-Ou
Wang, Limei
Phan, Tin
Clohessy, John G.
Pandolfi, Pier Paolo
author_facet Zhang, Yang
Nguyen, Tuan M.
Zhang, Xiao-Ou
Wang, Limei
Phan, Tin
Clohessy, John G.
Pandolfi, Pier Paolo
author_sort Zhang, Yang
collection PubMed
description Short hairpin RNAs (shRNAs) are used to deplete circRNAs by targeting back-splicing junction (BSJ) sites. However, frequent discrepancies exist between shRNA-mediated circRNA knockdown and the corresponding biological effect, querying their robustness. By leveraging CRISPR/Cas13d tool and optimizing the strategy for designing single-guide RNAs against circRNA BSJ sites, we markedly enhance specificity of circRNA silencing. This specificity is validated in parallel screenings by shRNA and CRISPR/Cas13d libraries. Using a CRISPR/Cas13d screening library targeting > 2500 human hepatocellular carcinoma-related circRNAs, we subsequently identify a subset of sorafenib-resistant circRNAs. Thus, CRISPR/Cas13d represents an effective approach for high-throughput study of functional circRNAs. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13059-021-02263-9.
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spelling pubmed-78189372021-01-22 Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs Zhang, Yang Nguyen, Tuan M. Zhang, Xiao-Ou Wang, Limei Phan, Tin Clohessy, John G. Pandolfi, Pier Paolo Genome Biol Method Short hairpin RNAs (shRNAs) are used to deplete circRNAs by targeting back-splicing junction (BSJ) sites. However, frequent discrepancies exist between shRNA-mediated circRNA knockdown and the corresponding biological effect, querying their robustness. By leveraging CRISPR/Cas13d tool and optimizing the strategy for designing single-guide RNAs against circRNA BSJ sites, we markedly enhance specificity of circRNA silencing. This specificity is validated in parallel screenings by shRNA and CRISPR/Cas13d libraries. Using a CRISPR/Cas13d screening library targeting > 2500 human hepatocellular carcinoma-related circRNAs, we subsequently identify a subset of sorafenib-resistant circRNAs. Thus, CRISPR/Cas13d represents an effective approach for high-throughput study of functional circRNAs. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13059-021-02263-9. BioMed Central 2021-01-21 /pmc/articles/PMC7818937/ /pubmed/33478577 http://dx.doi.org/10.1186/s13059-021-02263-9 Text en © The Author(s) 2021 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Method
Zhang, Yang
Nguyen, Tuan M.
Zhang, Xiao-Ou
Wang, Limei
Phan, Tin
Clohessy, John G.
Pandolfi, Pier Paolo
Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs
title Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs
title_full Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs
title_fullStr Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs
title_full_unstemmed Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs
title_short Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs
title_sort optimized rna-targeting crispr/cas13d technology outperforms shrna in identifying functional circrnas
topic Method
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7818937/
https://www.ncbi.nlm.nih.gov/pubmed/33478577
http://dx.doi.org/10.1186/s13059-021-02263-9
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