Fine tuning gene expression levels in mammalian cells with engineered microRNA target sites

Thesis

Precise, analogue regulation of gene expression is critical for development, homeostasis and regeneration in mammals. In contrast, widely employed experimental and therapeutic approaches such as knock-in/out strategies are more suitable for binary control of gene activity, while RNA interference (RNAi) can lead to pervasive off-target effects and unpredictable levels of repression. In this thesis, I report on a method for precise control of gene expression levels in mammalian cells based on engineered, synthetic microRNA response elements (MREs). To develop this system, we first tested a small panel of rationally designed MREs with various mismatches to miR-17 or miR-21. These MRE variants exerted predictable, stepwise control over reporter gene expression in a human cell line. Next, to improve the precision of MRE-based gene tuning, we established a high-throughput sequencing approach for measuring the efficacy of thousands of miR-17 MRE variants in parallel. This allowed us to create a library of microRNA silencing-mediated fine-tuners (miSFITs) of varying strength that can be employed to control the expression of user specified genes. A selection of miSFITs enabled precise, predictable tuning of PD-1, a co-inhibitory receptor expressed on T-cells and an important target for anti-tumour immunotherapy. In addition to tuning a PD-1 transgene in Jurkat T-cells, we successfully introduced miSFITs into the endogenous PD-1 3’UTR in embryonic stem cells using the CRISPR/Cas9 system. To further demonstrate the value of this technology, we also used a panel of miSFITs to tune the expression of a tumour-associated antigen in a mouse melanoma model. This analysis revealed that antigen expression level is a key determinant of the anti-tumour immune response in vitro and in vivo. miSFITs are a powerful tool for modulating gene expression levels with applications in research and cellular engineering.

Authors: Michaels, Y

• DOI: 10.5287/ora-mqyrqqpxr
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Keywords: microRNA; Tumour immunology; Genome engineering; Synthetic biology
• Subjects: Molecular biology; Synthetic biology; Genome Engineering