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Nat Commun. 2019 Feb 18;10(1):818. doi: 10.1038/s41467-019-08777-y.

Precise tuning of gene expression levels in mammalian cells.

Author information

1
Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, OX3 9DS, UK.
2
MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine University of Oxford, Oxford, OX3 9DS, UK.
3
Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, UK.
4
Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Oxford, OX3 7FY, UK.
5
School of Biological Sciences, University of Auckland, Auckland, 1050, New Zealand.
6
Weatherall Institute of Molecular Medicine, MRC Molecular Haematology Unit, NIHR Oxford Biomedical Research Centre Programme, University of Oxford, Oxford, OX3 9DS, UK.
7
Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, OX3 9DS, UK. tudor.fulga@imm.ox.ac.uk.

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.

PMID:
30778069
PMCID:
PMC6379387
DOI:
10.1038/s41467-019-08777-y
[Indexed for MEDLINE]
Free PMC Article

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