Precise tuning of gene expression levels in mammalian cells

Yale S Michaels,Thomas A Milne,Marco Fritzsche,Mary K Thompson,Hilary M Sheppard,Huw Colin-York,Hector Barbosa,Mike B Barnkob,David J H F Knapp,Toni A Baeumler,Violaine Andre,Uzi Gileadi,Tudor A Fulga,Vincenzo Cerundolo

doi:10.1038/s41467-019-08777-y

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.

Highlights

IntroductionAnalogue regulation of gene expression is critical for cellular function in mammals
Precise, analogue regulation of gene expression is critical for cellular function in mammals
We designed a 23nt degenerate oligonucleotide pool with 91% complementarity to miR-17 and 3% of each alternative nucleotide at every position (Fig. 1a, Supplementary Figure 1). This oligo pool was cloned downstream of a fluorescent reporter (ECFP) in a mammalian expression plasmid and the ensuing microRNA response elements (MREs) variant library was transfected into HEK-293T cells that endogenously express miR-17

Summary

Introduction

Analogue regulation of gene expression is critical for cellular function in mammals. 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 identify nucleotides that differentially impact repression and determine that quantifying transcript abundance is sufficient to accurately predict protein output levels We use this information to create a panel of miRNA silencing-mediated fine-tuners (miSFITs) and apply them to precisely modulate the expression levels of multiple genes including PD-1, a T-cell co-inhibitory receptor and a target for cancer immunotherapy. We use CRISPR/Cas[9] to integrate miSFITs into the 3′UTR of the key tumour suppressor gene BRCA117, demonstrating that it is possible to achieve geneticallyencoded fine tuning of endogenous gene expression levels in mammalian cells

Objectives

Methods

Results

Conclusion