ENGINEERING OF A MOUSE MODEL OF CHOLINE ACETYLTRANSFERASE DEFICIENCY USING CRISPR/CAS9 GENE EDITING Scientific Abstract: Choline acetyltransferase (ChAT) is the enzyme that resynthesizes the neurotransmitter acetylcholine, from acetyl-CoA and choline at the soma of multiple types of neurons of the peripheral and central nervous system (CNS). Mutations in the ChAT gene (CHAT) result in a potentially fatal form of human congenital myasthenic syndrome (CMS) referred to as CMS associated with episodic apneas. Chat knockout (KO) mice are immobile and die at birth, and conditional Chat KO (Chatflox/flox/Cre-ERT2) mice die a few weeks after the induction of Chat ablation by an intraperitoneal injection of tamoxifen. However, the lethal Chatflox/flox/Cre-ERT2 phenotype can be rescued in most cases by a single intracranial injection at the first day of life of 2x1013 vg/kg adeno-associated virus type 9 (AAV9) carrying the human CHAT gene (AAV9-CHAT). Furthermore, injected mice can reach adult life without developing obvious weakness. While these results are encouraging, the widespread depletion of Chat induced in the conditional knock out model is not an adequate model of the human disease, nor the injection of the therapeutic agent at the first day of life reflects the most likely time of therapeutic interventions in humans. Thus, to refine the model of CMS due to ChAT deficiency, we plan to create knock in (KI) mice homozygous for two previously identified human CHAT mutations, p.Val136 Met and p.Arg207His, using CRISPR/Cas9 gene editing. We will then attempt to correct the phenotype of ChatKI/KI mice by injecting intracisternally AAV9-CHAT during early postnatal life. The overall goal of the project is to generate an animal model that will reproduce as faithfully as possible the human disease and to conduct a pilot feasibility study on the efficacy of AAV9-CHAT mediated gene therapy in this newly developed animal model of CMS caused by CHAT mutations.