Institute of Genomics, School of Biomedical Sciences, Huaqiao University, 201 Pan-Chinese S & T Building, 668 Jimei Road, Xiamen, 361021, China.
Sci Rep. 2020 Feb 4;10(1):1794. doi: 10.1038/s41598-020-58104-5.
Long non-coding (lnc) RNAs represent a fascinating class of transcripts that remains highly controversial mainly due to ambiguity surrounding overall biological relevance of these RNAs. Multitude of reverse genetics studies showing functionality of lncRNAs are unfortunately based on assays that are either plagued by non-specific effects and/or cannot unambiguously assign observed phenotypes to the transcript per se. Here, we show application of the novel CRISPR/Cas13 RNA knockdown system that has superior specificity compared to other transcript-targeting knockdown methods like RNAi. We applied this method to a novel widespread subclass of nuclear lncRNAs - very long intergenic non-coding (vlinc) RNAs - in a high-throughput phenotypic assay based on survival challenge in response to anticancer drug treatments. We used multiple layers of controls including mismatch control for each targeting gRNA to ensure uncovering true phenotype-transcript relationships. We found evidence supporting importance for cellular survival for up to 60% of the tested protein-coding mRNAs and, importantly, 64% of vlincRNAs. Overall, this study demonstrates utility of CRISPR/Cas13 as a highly sensitive and specific tool for reverse genetics study of both protein-coding genes and lncRNAs. Furthermore, importantly, this approach provides evidence supporting biological significance of the latter transcripts in anticancer drug response.
长非编码（lnc）RNA 代表了一类引人入胜的转录本，主要由于这些 RNA 的整体生物学相关性存在模糊性，因此仍然存在很大争议。大量显示 lncRNA 功能的反向遗传学研究都是基于存在非特异性效应和/或不能将观察到的表型明确归因于转录本本身的测定方法。在这里，我们展示了新型 CRISPR/Cas13 RNA 敲低系统的应用，该系统与其他靶向转录物的敲低方法（如 RNAi）相比具有更高的特异性。我们将该方法应用于一种新型的广泛存在的核 lncRNA——非常长的基因间非编码（vlinc）RNA，在基于抗癌药物治疗的生存挑战的高通量表型测定中。我们使用了多层控制，包括针对每个靶向 gRNA 的错配对照，以确保揭示真实的表型-转录物关系。我们发现证据支持多达 60%的测试蛋白编码 mRNA 和重要的 64%的 vlincRNA 对细胞生存很重要。总体而言，这项研究证明了 CRISPR/Cas13 作为蛋白编码基因和 lncRNA 反向遗传学研究的高度敏感和特异性工具的实用性。此外，重要的是，这种方法为后者在抗癌药物反应中的生物学意义提供了证据支持。
