Bioluminescence resonance energy transfer (BRET) is a technology often used to probe the proximity of molecules within live cells. BRET assays have many applications including monitoring protein-protein interactions, examining receptor-ligand interactions, and screening for drug candidates. The versatility of BRET has encouraged researchers to discover new luminescent tools, including luciferase enzymes, and to enhance their performance. The recent development of nanoBRETTM is a case in point. Another direction to drive progress is to improve the tools used side by side with nanoBRET. One limitation of existing assays is that the fusion proteins needed for luminescence assays are generated by exogenous expression. In a recent development, nanoBRET was used with a CRISPR/Cas9 system to allow for endogenous expression of donor-fused proteins. CRISPR/Cas9 systems have revolutionised genome engineering by making it easier to make precise, targeted changes to DNA. Here we take a closer look at this advance, describe an application used to study G protein-coupled receptors, and discuss how this type of strategy can be used more widely to perform assays under more physiologically relevant conditions.
The Fundamentals of BRET
BRET is a type of non-radiative transfer of energy between a bioluminescent donor and a fluorescent acceptor (Figure 1). In BRET, the bioluminescent donor is typically a luciferase enzyme that comes into proximity with a fluorophore (usually less than 10 nm apart). One advantage of BRET is that it allows for real time monitoring of biological processes in living cells without the need for external light sources.
BRET assays typically use an ATP-dependent luciferase (often Rluc8) as the energy donor, a green fluorescent protein variant as the energy acceptor (e.g. Venus), and coelenterazine h or coelenterazine 400a as substrate for the luciferase enzyme. However, the availability of the new luciferase
reporter nanoluciferase (Nluc) and its substrate furimazine has resulted in the development of nanoBRET.1 NanoBRET offers enhanced sensitivity and precision due to a smaller and brighter bioluminescent donor compared to traditional luciferases like Renilla luciferase (Rluc8). Nluc is a small 19-kDa luciferase isolated from the deep-sea shrimp Oplophorus gracilirostris. It emits a bright, stable luminescence in a narrow spectrum. It offers higher sensitivity than Rluc8 and requires lower levels of expression which makes it amenable for a wide range of assays. The increased sensitivity of nanoBRET offers opportunities on other fronts to take advantage of further innovation. The ability to fuse a donor luciferase to endogenous proteins, for example, is now facilitated by different ways to engineer genomes including the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system.
The Benefits of CRISPR/Cas9 Genome Engineering
The discovery of the CRISPR/Cas9 system as a method for genome editing has revolutionised genome engineering.2 This gene-editing system, akin to a molecular scissors, allows for genetic modifications to be introduced into genomes with precision, speed and efficiency. The Cas9 protein is an endonuclease enzyme that cuts DNA at specific locations. The system also includes a guide RNA that is a short synthetic RNA composed of two parts: a scaffold sequence that binds to Cas9 and a spacer sequence complementary to the target DNA sequence. The spacer is crucial to direct Cas9 to the precise location where the modifications need to be made. The result is precise, targeted genome editing that is not only easy to use but which is also cost effective.
There are two different ways that CRISPR can introduce modifications depending on the type of repair that ensues after a double-stranded break is introduced into DNA. The differences arise depending on whether the breaks are repaired by error-prone non-homology end joining (NHEJ) or homology- directed repair (HDR). NHEJ results in random insertion-deletions (indels) and gene disruption at the target site. HDR can be harnessed to introduce a specific DNA template (single- or double-stranded) at the target site for precise gene editing.
