A CRISPR/Cas13-based approach demonstrates biological relevance of vlinc class of long non-coding RNAs in anticancer drug response.
A CRISPR/Cas13-based approach demonstrates biological relevance of vlinc class of long non-coding RNAs in anticancer drug response.
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基于 CRISPR/Cas13 的方法证明了 vlinc 类长非编码 RNA 在抗癌药物反应中的生物学相关性。
DOI：
10.1038/s41598-020-58104-5
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发表时间：
2020
影响因子：
4.6
通讯作者：
Kapranov Philipp
中科院分区：
文献类型：
--
作者：
Xu Dongyang;Cai Ye;Tang Lu;Han Xueer;Gao Fan;Cao Huifen;Qi Fei;Kapranov Philipp
长链非编码（lnc） rna代表了一类令人着迷的转录本，但由于围绕这些rna的总体生物学相关性的模糊性，仍然存在高度争议。不幸的是，许多显示lncrna功能的反向遗传学研究都是基于非特异性效应和/或不能明确地将观察到的表型分配给转录子本身的分析。在这里，我们展示了新型CRISPR/Cas13 RNA敲低系统的应用，与其他转录靶向敲低方法（如RNAi）相比，该系统具有更高的特异性。我们将这种方法应用于一种新的广泛分布的核lncrna亚类-非常长基因间非编码（vlinc） rna -在高通量表型分析中，基于抗癌药物治疗的生存挑战。我们使用了多层控制，包括对每个靶向gRNA的错配控制，以确保发现真正的表型-转录物关系。我们发现有证据支持高达60%的测试蛋白编码mrna和64%的vlincRNAs对细胞存活的重要性。总的来说，本研究证明了CRISPR/Cas13作为蛋白质编码基因和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，该方法提供了支持后者转录物在抗癌药物反应中的生物学意义的证据。
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 transcriptper 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.