Precise tuning of gene expression levels in mammalian cells.
3' Untranslated Regions
Animals
B7-H1 Antigen
/ genetics
CRISPR-Cas Systems
Gene Expression Regulation
/ genetics
Genes, BRCA1
Genetic Techniques
HEK293 Cells
High-Throughput Nucleotide Sequencing
/ methods
Humans
Melanoma, Experimental
/ genetics
Mice, Inbred C57BL
MicroRNAs
/ genetics
Ovalbumin
/ genetics
Recombinant Proteins
/ genetics
Response Elements
Xenograft Model Antitumor Assays
Journal
Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555
Informations de publication
Date de publication:
18 02 2019
18 02 2019
Historique:
received:
20
12
2018
accepted:
29
01
2019
entrez:
20
2
2019
pubmed:
20
2
2019
medline:
16
4
2019
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 30778069
doi: 10.1038/s41467-019-08777-y
pii: 10.1038/s41467-019-08777-y
pmc: PMC6379387
doi:
Substances chimiques
3' Untranslated Regions
0
B7-H1 Antigen
0
CD274 protein, human
0
MicroRNAs
0
Recombinant Proteins
0
Ovalbumin
9006-59-1
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
818Subventions
Organisme : Medical Research Council
ID : G0902418
Pays : United Kingdom
Organisme : Medical Research Council
ID : MC_UU_00008/1
Pays : United Kingdom
Organisme : Medical Research Council
ID : MC_UU_00016/6
Pays : United Kingdom
Organisme : Medical Research Council
ID : MC_UU_12009/6
Pays : United Kingdom
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