Precise tuning of gene expression levels in mammalian cells

被引：47

作者：

Michaels, Yale S. ^{[1
]}

Barnkob, Mike B. ^{[2
]}

Barbosa, Hector ^{[1
]}

Baeumler, Toni A. ^{[1
]}

Thompson, Mary K. ^{[3
]}

Andre, Violaine ^{[2
]}

Colin-York, Huw ^{[2
]}

Fritzsche, Marco ^{[2
,4
]}

Gileadi, Uzi ^{[2
]}

Sheppard, Hilary M. ^{[5
]}

Knapp, David J. H. F. ^{[1
]}

Milne, Thomas A. ^{[6
]}

Cerundolo, Vincenzo ^{[2
]}

Fulga, Tudor A. ^{[1
]}

机构：

[1] Univ Oxford, Weatherall Inst Mol Med, Radcliffe Dept Med, Oxford OX3 9DS, England

[2] Univ Oxford, MRC Human Immunol Unit, Weatherall Inst Mol Med, Oxford OX3 9DS, England

[3] Univ Oxford, Dept Biochem, Oxford OX1 3QU, England

[4] Kennedy Inst Rheumatol, Nuffield Dept Orthopaed Rheumatol & Musculoskelet, Oxford OX3 7FY, England

[5] Univ Auckland, Sch Biol Sci, Auckland 1050, New Zealand

[6] Univ Oxford, Weatherall Inst Mol Med, MRC Mol Haematol Unit, NIHR Oxford Biomed Res Ctr Programme, Oxford OX3 9DS, England

来源：

NATURE COMMUNICATIONS | 2019年 / 10卷 / 1期

基金：

英国生物技术与生命科学研究理事会; 英国惠康基金; 英国工程与自然科学研究理事会; 英国医学研究理事会; 欧洲研究理事会;

关键词：

MICRORNA; BINDING; AFFINITY;

D O I：

10.1038/s41467-019-08777-y

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

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.

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