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

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

Abstract 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 o...

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Bibliographic Details
Main Authors: Yang Zhang, Tuan M. Nguyen, Xiao-Ou Zhang, Limei Wang, Tin Phan, John G. Clohessy, Pier Paolo Pandolfi
Format: Article
Language:English
Published: BMC 2021-01-01
Series:Genome Biology
Subjects:
circRNAs
CRISPR/Cas13d system
High-throughput screening
Online Access:https://doi.org/10.1186/s13059-021-02263-9
Description
Summary:Abstract 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.
ISSN:1474-760X

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