October 1, 2018 (Vol. 38, No. 17)
Andrea Krumm Ph.D. Applications Specialist BMG Labtech
Combined Progress: CRISPR-Cas9 Enables Endogenous Donor Expression and CLARIOstar Sensitive Detection
G-protein-coupled receptors (GPCRs) are estimated to be targeted by 35% of FDA-approved drugs and constitute the biggest protein group targeted by drugs.1 Not surprisingly, pharmaceutical research extensively studies GPCRs which transmit signals from the outside to the inside of a cell. It is not only of interest whether a compound binds a specific receptor to activate or inhibit it, but also which signaling is triggered and whether any further receptor regulation follows an initial binding event.
A popular tool to measure receptor binding is bioluminescence resonance energy transfer (BRET) in microplate format. The energy transfer takes place between the light emitted by a luciferase and a fluorophore only if both are in highest proximity. Typically, the receptor of interest is labelled with a luciferase that emits light when converting its substrate. Expression of the fusion protein is achieved by stably or transiently transfecting it into a cell line of interest leading to exogenous and, hence, non-physiologic receptor levels. The luciferase-labelled receptor acts as a donor for a fluorophore which is fused to an interaction partner. Only if that is bound to the receptor, the fluorophore accepts the energy coming from the receptor-bound luciferase and emits light. The ratio of emission light from acceptor fluorophore to luciferase signal reveals the interaction of both.
So far, the method was limited by exogenous donor expression and by the use of a single acceptor, limiting the analysis to one interaction. Following, it is explained how CRISPR-Cas9 gene editing was used to express the donor GPCR at endogenous level and how those cells assisted in monitoring receptor trafficking by using a dual-acceptor approach.
Endogenous Receptor Expression Is Sufficient for Monitoring Protein Interaction
The chemokine receptor type 4 (CXCR4) is a GPCR implicated in cancer progression and therefore is a target for cancer therapy.2 Upon binding its ligand CXCL12, CXCR4 is activated and then recruits β-arrestin2 (β-arr2). Subsequently, the receptor is internalized for either recycling back to the plasma membrane or lysosomal breakdown. A group of researchers from the Australian Harry Perkins Institute of Medical Research was interested in studying these mechanisms of CXCR4.
To this end, the group used CRISPR-Cas9 to insert the DNA coding for nanoluciferase (Nluc) into the endogenous locus of CXCR4 in HEK293 cells.3 This way, the resulting BRET donor (receptor + luciferase) was expressed at endogenous level. The Nluc was chosen as it is very bright and results in sufficient light emission despite low expression levels.
In an initial experiment, the CRISPR-Cas9–edited cells were tested regarding their suitability for BRET experiments. The CXCR4/Nluc expressing cells were transiently transfected with cDNA coding for a β-arr2/Venus BRET acceptor (Figure A). As soon as cells expressing BRET-donor and -acceptor were treated with CXCL12, the CRXCR4 ligand, the BRET ratio increased and reached its maximum approximately 90 s after treatment (Figure B). This is explained by the recruitment of β-arr2/Venus to CXCR4/Nluc, a well-established step in receptor-regulation. The resulting proximity of Nluc and Venus fluorophore allows energy transfer and increased emission of Venus and consequently a higher BRET ratio.
Dual BRET Receptor Approach Reports on Receptor Trafficking
After demonstrating that the BRET assay with endogenous levels of the donor works, the researchers went one step further: they asked whether it is possible to combine the CXCR4/Nluc BRET donor with two acceptor fluorophores exhibiting different spectral properties and different intracellular interactions sites. They hoped to enable the NanoBRET™ method to monitor receptor trafficking. The interaction partners the group chose were K-ras as a membrane marker and Rab4 as a marker for the early endosome where the receptor is prepared for recycling. K-ras was fused to the NanoBRET 618 acceptor emitting in the red range and Rab4 to Venus emitting in the green range. Both acceptors were transiently transfected into the CRISPR-Cas9 genome-edited cells expressing CXCR4/Nluc (Figure C).
From the analysis point of view, the dual acceptor approach is particularly challenging. First of all, emissions from three light-emitting molecules need to be separated: blue signal coming from the Nluc (donor), red signal (membrane marker, KRAS/NanoBRET 618), and green signal (early endosome marker, RaB4/Venus).
Microplate readers usually measure BRET with filter-based systems consisting of two filters: one for the luciferase and one for the acceptor fluorophore. This is clearly no option for the Australian researchers. Monochromators offer the possibility to measure at any and several wavelengths. However, they are mostly limited in sensitivity. As the dual acceptor BRET works with endogenous and low levels of the donor, a low signal is expected and the detection needs to be extremely sensitive.
The group found a solution in the CLARIOstar® microplate reader with a patented linear variable filter (LVF) monochromator. The unique monochromator uses variable filters for wavelength selection, which allows it to use broad bandwidths and provides better light transmission, both leading to the highest sensitivity of monochromator-based readers.
Using the CLARIOstar for detection of the dual acceptor BRET assay has proven successful: Cells expressing CXCR4/Nluc at endogenous level and the two acceptors, K-ras/NanoBRET 618 and Rab4/Venus, responded to treatment with the ligand CXCL12. The BRET ratio for the membrane marker K-ras decreased upon treatment, indicating dissociation from the membrane. In contrast, BRET ratio of the endosome marker Rab4 increased, which reports the shuttling of CXCR4 to the early endosome upon agonist treatment (Figure D).
CRISPR-Cas9 and State-of-the-Art Detection Technology Expand NanoBRET Technology
The novel CRISPR-Cas9 technique successfully fused the Nluc BRET donor to endogenously expressed CXCR4. Luminescence generated by the resulting protein-luciferase fusion was sufficient to monitor receptor-protein interactions as well as trafficking. The multiplex internalization assay depends on two acceptor fluorophores whose selective detection was rendered possible by the CLARIOstar’s monochromator.