The aim of the Testing System is to provide information about the gRNA ability to localize the target. Therefore, external and intrinsic factors affecting the CRISPR/Cas9 mechanism should be blocked, letting us relate the light measurement exclusively with the gRNA efficacy to find the target. Moreover, this system emulates the performance of the gRNA in the real variety which is desired to be improved. Thus, the fact of implementing the Testing System in Nicotiana benthamiana should not affect the reliability of the result, making it scalable and realistic.
The goal is to characterize the performance of our Testing System in Nicotiana benthamiana, studying the influence of different factors affecting each stage of the CRIPSR/Cas9 mechanism, which relies on
binding and unbinding reactions, gene expression regulation, diffusion of biochemical compounds inside the nucleus and even thermodynamics.
In this Modeling task, we firstly developed a mathematical model accounting all these processes, structured in the main steps of the performance of CRISPR/Cas9. Afterwards, we simulated different conditions that result in variations of our reporter: Luciferase.
Thus, we can analyze determinant factors affecting Testing System repeatability, reliability and robustness, which are mandatory achievements in order to spread our gRNA Testing System as a standard gRNA efficiency predictor.