The development of a guided inducible dCas9 system to block FKBP5 demethylation and the effect of this blockade on FKBP5 expression and the development of Post Traumatic Stress Disorder

FKBP5 is a gene that plays a crucial role in stress response and is known to be involved in stress-related psychiatric disorders, particularly Post Traumatic Stress Disorder (PTSD). PTSD is a devastating condition that has an adverse impact in Canada and across the world with an estimated lifetime prevalence of 3.9% in the general population and 5.6% amongst people who have been exposed to trauma. Women are also more likely to be affected than men, with rates almost twice as high. In Canada, it is estimated that 9.2% of the population will experience PTSD at some point in their lives. PTSD can be caused by exposure to a traumatic event or even indirectly, when one strongly identifies to someone who has been exposed to the triggering trauma. The body naturally responds to such events with fear and other physiological mechanisms, it is the “fight or flight response”. However, if the symptoms persist over time, it can lead to a diagnosis of PTSD. Although studies have suggested a correlation between the methylation state of FKBP5 and the likelihood of developing PTSD after early trauma, such as child abuse, the causal relationship between the methylation state of cytosine-phosphate-guanine-dinucleotides (CpGs) and long-term stress effects is still unknown. Our hypothesis is that if FKBP5 demethylation plays a causal role in the stress response, preventing it from happening would dampen the stress response. To investigate this, we developed an inducible dCas9 model that targets specific sites in FKBP5, which are believed to be involved in the gene's regulation and impact on stress response. We aim to test this system in HEK cells, confirm if early stress demethylates these CpG sites, determine when this occurs, and prevent it from happening using our dCas9 inducible system. By studying how this affects FKBP5 expression and the cell's response to stress, we can establish the causal role between methylation and stress response. Our mutagenesis and lentiviral experiments helped us develop the system, which we then infected into HEK 293T cells. We used Western blot to test the efficiency of the system. We successfully created a pool of inducible plasmids targeting 14 different CpG sites potentially important in FKBP5 regulation. We infected HEK 293T cells with some of these plasmids to test the system and were able to study the optimum conditions for induction and system efficiency. We also established a baseline methylation state/percentage of important CpG sites in HEK 293T cells. We have started developing a model with oxidized CpG sites in FKBP5 to test the system's efficiency on them. Our experiments show that we can create a fully adaptable and efficient inducible dCas9 system that targets specific sites for a certain amount of time, allowing steric inhibition of methylation or demethylation. This could lead to the development of new treatments and prevention strategies for PTSD and other psychiatric stress-related disorders

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