State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China.
CAS Key Laboratory of Computational Biology, CAS-MPG Partner Institute for Computational Biology, Shanghai Institute of Nutrition and Health, Shanghai Institutes for Biological Sciences, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China.
Nat Methods. 2021 Jan;18(1):51-59. doi: 10.1038/s41592-020-01011-4. Epub 2020 Dec 7.
Circular RNAs (circRNAs) produced from back-spliced exons are widely expressed, but individual circRNA functions remain poorly understood owing to the lack of adequate methods for distinguishing circRNAs from cognate messenger RNAs with overlapping exons. Here, we report that CRISPR-RfxCas13d can effectively discriminate circRNAs from mRNAs by using guide RNAs targeting sequences spanning back-splicing junction (BSJ) sites featured in RNA circles. Using a lentiviral library that targets sequences across BSJ sites of highly expressed human circRNAs, we show that a group of circRNAs are important for cell growth mostly in a cell-type-specific manner and that a common oncogenic circRNA, circFAM120A, promotes cell proliferation by preventing the mRNA for family with sequence similarity 120A (FAM120A) from binding the translation inhibitor IGF2BP2. Further application of RfxCas13d-BSJ-gRNA screening has uncovered circMan1a2, which has regulatory potential in mouse embryo preimplantation development. Together, these results establish CRISPR-RfxCas13d as a useful tool for the discovery and functional study of circRNAs at both individual and large-scale levels.
环状 RNA（circRNAs）由反向剪接的外显子产生，广泛表达，但由于缺乏区分具有重叠外显子的 circRNA 和同源信使 RNA 的适当方法，单个 circRNA 的功能仍知之甚少。在这里，我们报告说，CRISPR-RfxCas13d 可以通过使用靶向跨越 RNA 环中特征性的反向剪接连接（BSJ）位点的序列的向导 RNA，有效地将 circRNAs 与 mRNAs 区分开来。使用针对高度表达的人类 circRNAs 的 BSJ 位点的靶向序列的慢病毒文库，我们表明一组 circRNAs 对细胞生长很重要，主要是以细胞类型特异性的方式，并且常见的致癌 circRNA circFAM120A 通过防止家族与序列相似性 120A（FAM120A）的 mRNA 结合翻译抑制剂 IGF2BP2 来促进细胞增殖。RfxCas13d-BSJ-gRNA 筛选的进一步应用揭示了 circMan1a2，它在小鼠胚胎植入前发育中具有调节潜力。总之，这些结果确立了 CRISPR-RfxCas13d 作为一种在个体和大规模水平上发现和研究 circRNAs 的有用工具。
