Cancer Research Institute, Beth Israel Deaconess Cancer Center, Department of Medicine and Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02215, USA.
Ludwig Center at Harvard, Harvard Medical School, Boston, MA, 02215, USA.
Genome Biol. 2021 Jan 21;22(1):41. doi: 10.1186/s13059-021-02263-9.
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.
短发夹 RNA (shRNA) 可通过靶向反向剪接连接 (BSJ) 位点来耗尽 circRNA。然而，shRNA 介导的 circRNA 敲低与相应的生物学效应之间经常存在差异，质疑其稳健性。通过利用 CRISPR/Cas13d 工具并优化针对 circRNA BSJ 位点的单引导 RNA 的设计策略，我们显着提高了 circRNA 沉默的特异性。通过 shRNA 和 CRISPR/Cas13d 文库的平行筛选验证了这种特异性。使用靶向 > 2500 个与人类肝细胞癌相关的 circRNA 的 CRISPR/Cas13d 筛选文库，我们随后鉴定出一组索拉非尼耐药的 circRNA。因此，CRISPR/Cas13d 代表了一种用于功能性 circRNA 高通量研究的有效方法。
