Using nanoBRET and CRISPR/Cas9 to monitor proximity to a genome-edited protein in real-time

Abstract Bioluminescence resonance energy transfer (BRET) has been a vital tool for understanding G protein-coupled receptor (GPCR) function. It has been used to investigate GPCR-protein and/or -ligand interactions as well as GPCR oligomerisation. However the utility of BRET is limited by the requir...

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Autores principales:	Carl W. White, Hannah K. Vanyai, Heng B. See, Elizabeth K. M. Johnstone, Kevin D. G. Pfleger
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/7b3fcf6037e049f69f6a0bb8d24be1b0
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spelling	oai:doaj.org-article:7b3fcf6037e049f69f6a0bb8d24be1b02021-12-02T15:06:08ZUsing nanoBRET and CRISPR/Cas9 to monitor proximity to a genome-edited protein in real-time10.1038/s41598-017-03486-22045-2322https://doaj.org/article/7b3fcf6037e049f69f6a0bb8d24be1b02017-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-03486-2https://doaj.org/toc/2045-2322Abstract Bioluminescence resonance energy transfer (BRET) has been a vital tool for understanding G protein-coupled receptor (GPCR) function. It has been used to investigate GPCR-protein and/or -ligand interactions as well as GPCR oligomerisation. However the utility of BRET is limited by the requirement that the fusion proteins, and in particular the donor, need to be exogenously expressed. To address this, we have used CRISPR/Cas9-mediated homology-directed repair to generate protein-Nanoluciferase (Nluc) fusions under endogenous promotion, thus allowing investigation of proximity between the genome-edited protein and an exogenously expressed protein by BRET. Here we report BRET monitoring of GPCR-mediated β-arrestin2 recruitment and internalisation where the donor luciferase was under endogenous promotion, in live cells and in real time. We have investigated the utility of CRISPR/Cas9 genome editing to create genome-edited fusion proteins that can be used as BRET donors and propose that this strategy can be used to overcome the need for exogenous donor expression.Carl W. WhiteHannah K. VanyaiHeng B. SeeElizabeth K. M. JohnstoneKevin D. G. PflegerNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-14 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Carl W. White Hannah K. Vanyai Heng B. See Elizabeth K. M. Johnstone Kevin D. G. Pfleger Using nanoBRET and CRISPR/Cas9 to monitor proximity to a genome-edited protein in real-time
description	Abstract Bioluminescence resonance energy transfer (BRET) has been a vital tool for understanding G protein-coupled receptor (GPCR) function. It has been used to investigate GPCR-protein and/or -ligand interactions as well as GPCR oligomerisation. However the utility of BRET is limited by the requirement that the fusion proteins, and in particular the donor, need to be exogenously expressed. To address this, we have used CRISPR/Cas9-mediated homology-directed repair to generate protein-Nanoluciferase (Nluc) fusions under endogenous promotion, thus allowing investigation of proximity between the genome-edited protein and an exogenously expressed protein by BRET. Here we report BRET monitoring of GPCR-mediated β-arrestin2 recruitment and internalisation where the donor luciferase was under endogenous promotion, in live cells and in real time. We have investigated the utility of CRISPR/Cas9 genome editing to create genome-edited fusion proteins that can be used as BRET donors and propose that this strategy can be used to overcome the need for exogenous donor expression.
format	article
author	Carl W. White Hannah K. Vanyai Heng B. See Elizabeth K. M. Johnstone Kevin D. G. Pfleger
author_facet	Carl W. White Hannah K. Vanyai Heng B. See Elizabeth K. M. Johnstone Kevin D. G. Pfleger
author_sort	Carl W. White
title	Using nanoBRET and CRISPR/Cas9 to monitor proximity to a genome-edited protein in real-time
title_short	Using nanoBRET and CRISPR/Cas9 to monitor proximity to a genome-edited protein in real-time
title_full	Using nanoBRET and CRISPR/Cas9 to monitor proximity to a genome-edited protein in real-time
title_fullStr	Using nanoBRET and CRISPR/Cas9 to monitor proximity to a genome-edited protein in real-time
title_full_unstemmed	Using nanoBRET and CRISPR/Cas9 to monitor proximity to a genome-edited protein in real-time
title_sort	using nanobret and crispr/cas9 to monitor proximity to a genome-edited protein in real-time
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/7b3fcf6037e049f69f6a0bb8d24be1b0
work_keys_str_mv	AT carlwwhite usingnanobretandcrisprcas9tomonitorproximitytoagenomeeditedproteininrealtime AT hannahkvanyai usingnanobretandcrisprcas9tomonitorproximitytoagenomeeditedproteininrealtime AT hengbsee usingnanobretandcrisprcas9tomonitorproximitytoagenomeeditedproteininrealtime AT elizabethkmjohnstone usingnanobretandcrisprcas9tomonitorproximitytoagenomeeditedproteininrealtime AT kevindgpfleger usingnanobretandcrisprcas9tomonitorproximitytoagenomeeditedproteininrealtime
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Using nanoBRET and CRISPR/Cas9 to monitor proximity to a genome-edited protein in real-time

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