Optimizing CRISPR/Cas9 interactions for therapeutic approaches requires that the binding affinities of diverse RNA constructs to Cas9 be accurately determined. The classical methods used to determine these binding affinities include immunoprecipitation or electrophoretic mobility shift assays.
With these classical methods, there are some downfalls, which include:
1. The amount of time they take
2. The use of hazardous materials
3. The inability to reach a chemical equilibrium
Scientists at Axolabs had to find a technology that could overcome these problems when they needed to measure the binding affinities of a broad range of guide RNAs for Cas9. Using MST technology and the Monolith, the team was able to produce highly sensitive results in less than 2 minutes with minuscule amounts (pM-nM range) of non-hazardous fluorescent-labeled proteins or oligonucleotides. With MST they were even able to detect the affinity of the protein/RNA complex in equilibrium, in free solution, which is yet another advantage over these other classical methods.