Precise tuning of gene expression levels in mammalian cells

Yale S. Michaels, Mike B. Barnkob, Hector Barbosa, Toni A. Baeumler, Mary K. Thompson, Violaine Andre, Huw Colin-York, Marco Fritzsche, Uzi Gileadi, Hilary M. Sheppard, David J. H. F. Knapp, Thomas A. Milne, Vincenzo Cerundolo and Tudor A. Fulga ()
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Yale S. Michaels: University of Oxford
Mike B. Barnkob: Weatherall Institute of Molecular Medicine University of Oxford
Hector Barbosa: University of Oxford
Toni A. Baeumler: University of Oxford
Mary K. Thompson: University of Oxford
Violaine Andre: Weatherall Institute of Molecular Medicine University of Oxford
Huw Colin-York: Weatherall Institute of Molecular Medicine University of Oxford
Marco Fritzsche: Weatherall Institute of Molecular Medicine University of Oxford
Uzi Gileadi: Weatherall Institute of Molecular Medicine University of Oxford
Hilary M. Sheppard: University of Auckland
David J. H. F. Knapp: University of Oxford
Thomas A. Milne: University of Oxford
Vincenzo Cerundolo: Weatherall Institute of Molecular Medicine University of Oxford
Tudor A. Fulga: University of Oxford

Nature Communications, 2019, vol. 10, issue 1, 1-12

Abstract: Abstract Precise, analogue regulation of gene expression is critical for cellular function 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. Here we report on a method for precise control of gene expression levels in mammalian cells using engineered microRNA response elements (MREs). First, we measure the efficacy of thousands of synthetic MRE variants under the control of an endogenous microRNA by high-throughput sequencing. Guided by this data, we establish a library of microRNA silencing-mediated fine-tuners (miSFITs) of varying strength that can be employed to precisely control the expression of user-specified genes. We apply this technology to tune the T-cell co-inhibitory receptor PD-1 and to explore how antigen expression influences T-cell activation and tumour growth. Finally, we employ CRISPR/Cas9 mediated homology directed repair to introduce miSFITs into the BRCA1 3′UTR, demonstrating that this versatile tool can be used to tune endogenous genes.

Date: 2019
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DOI: 10.1038/s41467-019-08777-y

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