Anin vivostrategy for knockdown of circular RNAs

Abstract

Exonic circular RNAs (circRNAs) are highly abundant and evolutionarily conserved 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 miRNA-derived shRNAs targeted to the circRNAspecific back-splice junction to specifically downregulate the circRNA. We utilized this methodology to downregulate five circRNAs that are highly expressed in the fly nervous system. There were no effects on levels of the linear RNA 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, we found that 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 cell levels.

Circular RNA in disease: Basic properties and biomedical relevance

“…CircRNAs play pathogenic or suppressive roles and might serve as therapeutic targets in various diseases. Strategies, such as RNAi, ASO, and CRISPR‐mediated transcript knockdown (Abudayyeh et al, 2017; Özcan et al, 2021; Pamudurti et al, 2020; Song, Jia, et al, 2021; You et al, 2021), can be designed to reduce circRNA expression in specific tissues, and there are a few of studies supporting these new therapeutic approaches: (i) intratumor injection of the siRNA against circNRIP1 remarkably suppressed gastric cancer growth in mouse models (X. Zhang, Wang, et al, 2019); (ii) In vivo tail vein injection of the ASO targeting circLONP2 dramatically reduced the penetrance of metastasis to foreign organs in colorectal carcinoma (K. Han, Wang, et al, 2020b); (iii) genetically engineered circBoule knockout mice and fruit flies, which exhibit reduced male fertility in response to stressed conditions, have been successfully constructed by depleting the flanking introns of the circularizing exons through the CRISPR‐Cas 9 system (Gao et al, 2020).…”

The Secret Garden of Neuronal circRNAs

“…Two recent studies provided new approaches to KD circRNAs in vivo in animal models [ 74 , 75 ]. Pamudurti et al generated a Drosophila-specific shRNA vector based on the miR-1 precursor, expressing siRNAs directed against the BSJ of selected circRNAs [ 74 ]. The shRNA expression is temporally and spatially controlled by means of the GAL4/UAS expression system.…”