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Precise tuning of gene expression levels in mammalian cells

Author

Listed:
  • 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
    Rheumatology and Musculoskeletal Sciences)

  • 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)

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.

Suggested Citation

  • 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 , 2019. "Precise tuning of gene expression levels in mammalian cells," Nature Communications, Nature, vol. 10(1), pages 1-12, December.
    • Handle: RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-019-08777-y
    DOI: 10.1038/s41467-019-08777-y
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    Cited by:

    1. Stephane De Cesco & John B Davis & Paul E Brennan, 2020. "TargetDB: A target information aggregation tool and tractability predictor," PLOS ONE, Public Library of Science, vol. 15(9), pages 1-12, September.

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