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...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2021-01-01
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| Series: | Genome Biology |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s13059-021-02263-9 |
| _version_ | 1818325718674702336 |
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| author | Yang Zhang Tuan M. Nguyen Xiao-Ou Zhang Limei Wang Tin Phan John G. Clohessy Pier Paolo Pandolfi |
| author_facet | Yang Zhang Tuan M. Nguyen Xiao-Ou Zhang Limei Wang Tin Phan John G. Clohessy Pier Paolo Pandolfi |
| author_sort | Yang Zhang |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-13T11:48:56Z |
| format | Article |
| id | doaj.art-ddef8dbdffe34aa79a6e85564b8bfeab |
| institution | Directory Open Access Journal |
| issn | 1474-760X |
| language | English |
| last_indexed | 2024-12-13T11:48:56Z |
| publishDate | 2021-01-01 |
| publisher | BMC |
| record_format | Article |
| series | Genome Biology |
| spelling | doaj.art-ddef8dbdffe34aa79a6e85564b8bfeab2022-12-21T23:47:25ZengBMCGenome Biology1474-760X2021-01-0122112210.1186/s13059-021-02263-9Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAsYang Zhang0Tuan M. Nguyen1Xiao-Ou Zhang2Limei Wang3Tin Phan4John G. Clohessy5Pier Paolo Pandolfi6Cancer Research Institute, Beth Israel Deaconess Cancer Center, Department of Medicine and Pathology, Beth Israel Deaconess Medical Center, Harvard Medical SchoolCancer Research Institute, Beth Israel Deaconess Cancer Center, Department of Medicine and Pathology, Beth Israel Deaconess Medical Center, Harvard Medical SchoolProgram in Bioinformatics and Integrative Biology, University of Massachusetts Medical SchoolCancer Research Institute, Beth Israel Deaconess Cancer Center, Department of Medicine and Pathology, Beth Israel Deaconess Medical Center, Harvard Medical SchoolCancer Research Institute, Beth Israel Deaconess Cancer Center, Department of Medicine and Pathology, Beth Israel Deaconess Medical Center, Harvard Medical SchoolCancer Research Institute, Beth Israel Deaconess Cancer Center, Department of Medicine and Pathology, Beth Israel Deaconess Medical Center, Harvard Medical SchoolCancer Research Institute, Beth Israel Deaconess Cancer Center, Department of Medicine and Pathology, Beth Israel Deaconess Medical Center, Harvard Medical SchoolAbstract 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.https://doi.org/10.1186/s13059-021-02263-9circRNAsCRISPR/Cas13d systemHigh-throughput screening |
| spellingShingle | 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 circRNAs CRISPR/Cas13d system High-throughput screening |
| 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 | circRNAs CRISPR/Cas13d system High-throughput screening |
| url | https://doi.org/10.1186/s13059-021-02263-9 |
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