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

Yang Zhang, Tuan M Nguyen, Xiao-Ou Zhang, Limei Wang, Tin Phan, John G Clohessy, Pier Paolo Pandolfi

Genome biology 2021 Jan 21

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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.

Citation

Yang Zhang, Tuan M Nguyen, Xiao-Ou Zhang, Limei Wang, Tin Phan, John G Clohessy, Pier Paolo Pandolfi. Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs. Genome biology. 2021 Jan 21;22(1):41