Team:TU-Eindhoven/Read-out
We tested our designed heterodimers on functionality using NanoBiT assays and designed a CRISPR/Cas9 assay.
The NanoBiT assays were designed to test if our newly created scaffolds are functional heterodimers, and utilizes split NanoLuc luciferase. Split NanoLuc (also called NanoBiT) are two non-identical fragments which on their own are inactive, but after dimerization they form an active NanoLuc luciferase system, which emits light when a luminogenic substrate (furimazine) is present, see figure 1.
We used this feature to test whether our modified CT52 proteins bind to their complementary binding pocket on a monomer by fusing the NanoBiT fragments to the CT52 proteins using a flexible GGS10 linker: this is a 10 times repeating Glycine-Glycine-Serine sequence. The fragments of the split NanoLuc are called LargeBiT and SmallBiT. We worked with several variants of CT52, which we designed using the Rosetta software package (see the modeling section). In theory, the CT52-LargeBiT and CT52-SmallBiT will assemble on the heterodimeric scaffold under influence of fusicoccin, resulting in dimerization of the split NanoLuc fragments which leads to an emission of bioluminescence light with a wavelength of 460 nm. With this feature we can determine if the two split NanoLucs have assembled, thus giving information about the functionality and orthogonality of the scaffold.
The CRISPR/Cas9 assay is designed in order to test the viability of the application. The tested application is a fusicoccin regulated CRISPR/Cas9 system utilizing split Cas9. Under influence of fusicoccin both CT52 will assemble on the heterodimeric scaffold, leading to the dimerization of the split Cas9 fragments forming an active Cas9 nuclease. The Cas9 nuclease works by binding to a RNA strand which functions as a guide. When the RNA binds to a complementary piece of double stranded DNA, the Cas9 nuclease induces a double stranded break at that specific location, see figure 2. To check whether the assay has worked properly, agarose gel electrophoreses can be performed in order to see the specific base lengths of the cutted DNA strand.
