Characterization of Cas9-Mediated microRNA Knockout
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MicroRNAs are short regulatory RNAs that primarily operate at the posttranscriptional level, acting as part of the RISC complex to destabilize mRNA prior to, or during, translation. MicroRNAs are subject to several levels of processing by Drosha and Dicer prior to achievement of the active mature form, this processing being highly dependent on the secondary structure of the microRNA transcript. As such, microRNA biogenesis is highly sensitive to mutations. In this study, it was attempted to optimize Cas9-mediated genome editing techniques for microRNA knockout for two hematopoietic microRNAs, miR-142 and miR-223. While it has been previously demonstrated that Cas9 is capable of inhibiting microRNA biogenesis, the results have been characterized by variable and low-level efficiencies currently inherent to the system. This study attempted to rectify this shortcoming by bombarding the small pre-miR transcript with a combination of multiple independently targeted Cas9 complexes, using lentiviral delivery methods, measuring for evidence of impaired microRNA biogenesis by real-time quantitative PCR (RT-qPCR). The results suggest that there may be specific regions of the microRNA site in which mutations are more deleterious to function, but that further study is required. Additionally, it was determined that the use of multiple sgRNAs is not experimentally useful, proving unable to and in some cases, an impediment to successful microRNA knockout.
Morin, Andrew (2015). Characterization of Cas9-Mediated microRNA Knockout. Master's thesis, Texas A & M University. Available electronically from