Optimized CRISPR-RfxCas13d system for RNA targeting in zebrafish embryos.
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ABSTRACT:
SUBMITTER: Hernandez-Huertas L
PROVIDER: S-EPMC8715325 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Circular RNAs (circRNAs) are widely expressed, but their functions remain largely unknown. To study circRNAs in a high-throughput manner, short hairpin RNA (shRNA) screens1 have recently been used to deplete circRNAs by targeting their unique back-splicing junction (BSJ) sites. Here, we report frequent discrepancies between shRNA-mediated circRNA knockdown efficiency and the corresponding biological effect, raising pressing concerns about the robustness of shRNA screening for circRNA functional characterization. To address this issue, we leveraged the CRISPR/Cas13d system2 for functional study of circRNAs by optimizing the strategy for designing single guide RNAs to deplete circRNAs. We then performed shRNA and CRISPR/Cas13d parallel screenings and demonstrated that shRNA-mediated circRNA screening yielded a high rate of false positive phenotypes. Furthermore, using a CRISPR/Cas13d screening library targeting over 2,500 human hepatocellular carcinomas (HCC) related circRNAs, we identified a group of circRNAs, whose inhibition increased the therapeutic efficacy of sorafenib, an approved HCC drug. Collectively, these data demonstrate that CRISPR/Cas13d system is an effective approach to study the function of circRNAs in a high-throughput manner.
2020-12-06 | GSE162720 | GEO