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 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.

Optimised CRISPR-Cas9 mediated single molecule imaging for accurate quantification through endogenous expression

“…Immediately preceding transfection, cells were washed twice with sterile PBS before incubation in Optimem. The px459 guide and Cas9-puro expressing plasmid was used (Addgene plasmid #62988 was a gift from Feng Zheng) to introduce the previously reported TubA1B targeting guide C or CXCR4 targeting guide with Cas9 elements to transfected cells (15,40) . Suspension cells were transfected using the Neon electroporation system (Thermo).…”

“…Validated plasmids were amplified. Donor plasmids for CXCR4 genome engineering were generated by sub-cloning codon-optimised, mEos 4b, mEos 4b and HaloTag as well as mEGFP synthesised as doubled stranded DNA gBlocks (Integrated DNA Technologies) into the CXCR4 donor vector described previously using XhoI and XbaI restriction enzymes (40).…”

Fluorescent ligands: Bringing light to emerging GPCR paradigms

“…The advent of CRISPR/Cas9‐mediated genome engineering (Cong et al, ) has been a major breakthrough in cell biology. CRISPR/Cas9 has enabled the genetic modification of endogenously expressed proteins, including appending bioluminescent or fluorescent tags to the C‐terminus of endogenous GPCRs (Figure a; White, Vanyai, See, Johnstone, & Pfleger, ). White, Johnstone, See, and Pfleger () used CRISPR/Cas9 to generate an N‐terminally NanoLuc TM ‐tagged adenosine A 2B receptor and performed NanoBRET ligand binding in HEK293 cells.…”

Ligand-directed covalent labelling of a GPCR with a fluorescent tag

“…Each of these technologies has their limitations and are not readily transferrable to endogenously expressing systems where GPCRs are expressed at low levels. Gene editing techniques, such as CRISPR/Cas9 2 , have allowed labelling of proteins 3 , including GPCRs 4,5 , at endogenous levels but the fluorophore used can dramatically alter expression levels 6 . An orthogonal approach to protein labelling uses ligand-directed chemistry whereby connecting a fluorophore via a highly reactive, electrophilic linker to a ligand that binds to the protein of interest.…”