Exonic circular RNAs (circRNAs) are highly abundant RNAs generated mostly from exons of protein-coding genes. Assaying the functions of circRNAs is not straightforward as common approaches for circRNA depletion tend to also alter the levels of mRNAs generated from the hosting gene. Here we describe a methodology for specific knockdown of circRNAs in vivo with tissue and cell resolution. We also describe an experimental and computational platform for determining the potential off-target effects as well as for verifying the obtained phenotypes. Briefly, we utilize shRNAs targeted to the circRNA-specific back-splice junction to specifically downregulate the circRNA. We utilized this methodology to downregulate five circRNAs that are highly expressed in Drosophila. There were no effects on the levels of their linear counterparts or any RNA with complementarity to the expressed shRNA. Interestingly, downregulation of circCtrip resulted in developmental lethality that was recapitulated with a second shRNA. Moreover, downregulation of individual circRNAs caused specific changes in the fly head transcriptome, suggesting roles for these circRNAs in the fly nervous system. Together, our results provide a methodological approach that enables the comprehensive study of circRNAs at the organismal and cellular levels and generated for the first time flies in which specific circRNAs are downregulated.

外显子环状 RNA (circRNA) 是高度丰富的 RNA，主要由蛋白质编码基因的外显子产生。检测 circRNA 的功能并不简单，因为去除 circRNA 的常用方法往往也会改变宿主基因产生的 mRNA 水平。在这里，我们描述了一种通过组织和细胞分辨率在体内特异性敲除 circRNA 的方法。我们还描述了一个实验和计算平台，用于确定潜在的脱靶效应以及验证获得的表型。简而言之，我们利用靶向 circRNA 特异性反向剪接连接的 shRNA 来特异性下调 circRNA。我们利用这种方法下调了在果蝇中高度表达的五种 circRNA. 对其线性对应物或与表达的 shRNA 具有互补性的任何 RNA 的水平没有影响。有趣的是，circCtrip 的下调导致了发育致死率，并用第二个 shRNA 进行了概括。此外，单个 circRNA 的下调导致果蝇头部转录组的特定变化，表明这些 circRNA 在果蝇神经系统中的作用。总之，我们的结果提供了一种方法论方法，可以在生物体和细胞水平上对 circRNA 进行全面研究，并首次生成其中特定 circRNA 被下调的果蝇。