Abstract 18034: CRISPR/Cas9 Gene Editing Highlights the Prominent Role of Structure in Noncoding RNA Function: The Case of Clustered MicroRNAs in the Vasculature

Introduction: MicroRNAs (miRNAs) are noncoding RNAs often organized in clusters that emerged as critical regulators of the cardiovascular system. Hypothesis: We hypothesized that gene editing can elucidate the role of tertiary structure in regulating noncoding RNA function. Our studies focused on miRNA clusters. Methods and Results: Four miRNA clusters with critical functions in the vasculature, miR-497~195, miR-143~145, miR-17~92 and miR-106b~25 were assessed. To induce gene editing we generated mouse vascular smooth muscle cells stably expressing Cas9 nuclease. Cells were then transfected with an in vitro transcribed single guide RNA targeting miR-195, miR-145, miR-18a and miR-25 stem loop, respectively. T7 endonuclease I assay and Sanger sequencing confirmed efficient editing. QPCR demonstrated effective decrease of the targeted miRNAs. Intriguingly, in the case miR-195 no effect on miR-15a and miR-16, two highly expressed miRNAs belonging to the same miRNA family was observed. On the contrary, the expression of miR-497 was decreased in edited cells while no gene editing was detected in the miR-497 genomic locus. Of note, miR-195 and miR-497 form a miRNA cluster and are co-transcribed as a primary miRNA. Computational simulation predicted that mutations of the miR-195 stem loop led to changes in the tertiary structure of the primary miR-497~195 transcript that could affect its processing to mature miRNAs. Similar findings were obtained for the miR-143~145 cluster. Specific targeting of the miR-145 locus also inhibited the expression of miR-143 whereas no editing of the miR-143 locus was detected. Intriguingly, the expression of Carmen, a long noncoding RNA overlapping the miR-143~145 cluster that constitutes an independent transcription unit did not differ in miR-145 edited cells confirming that only the primary miRNA transcript is affected. On the other hand, in clusters miR-17~92 and miR-106b~25 differential miRNA expression was observed for the targeted miRNA, while the levels of the other miRNAs in the cluster were not affected. Conclusions: CRISPR/ Cas9 gene editing has revealed novel aspects of clustered miRNA regulation and underlined the importance of tertiary structure in noncoding RNA function.