CRISPR-based gene editing has the potential to revolutionize the treatment of genetic brain disorders. However, complications with brain delivery have limited the utility of CRISPR-based therapeutics. To address this critical need, we have developed a new gene editing delivery vehicle, termed CRISPR-PEG, which is composed of Cas9 RNP conjugated to polyethylene glycol (PEG). CRISPR-PEG has tremendous promise as a delivery vehicle because of its excellent biocompatibility, the well-established clinical track record of PEG, and its enhanced tissue diffusion capability in comparison to nanoparticles. Our preliminary results demonstrate that CRISPR-PEG delivers and edits neurons efficiently in the motor cortex or striatum in mice; after an intracranial injection, neurons were edited with a high specificity (45~85%). Notably, CRISPR-PEG also edited neurons in the olfactory bulb after intranasal administration. These exciting results demonstrate that CRISPR-PEG has great potential as bio-tool, and as a platform for developing therapeutics. In this proposal we will test our novel delivery vehicle CRISPR-PEG in fragile X syndrome (FXS). We have selected FXS as a test bed for CRISPR-PEG because it is the most common inherited cause of intellectual disability with no treatment available. In addition, FXS has a monogenic cause, namely expanded CGG repeats>200 and hypermethylation in the FMR1 promoter region, which causes silencing of the fragile X mental retardation 1 (FMR1) gene. Therefore, the central objectives of this proposal are (1) to test CRISPR-PEG in brain disorders by targeting FXS-associated genes, and (2) to develop new CRISPR-PEG variants with improved diffusion and efficiency. The central hypothesis is: the novel non-viral delivery vehicle CRISPR- PEG will deliver Cas9 RNPs into the brain, efficiently edit FXS-associated genes in neurons, and rescue mice from multiple FXS-associated phenotypes. The central objective will be accomplished by completing the following specific aims. Specific Aim 1. Knock down mGluR5 using CRISPR-PEG in the mouse model of FXS as proof of principle. Specific Aim 2. Reactivate FMR1 gene expression using CRISPR-PEG. Specific Aim 3. Develop CRISPR-PEGs that diffuse throughout the brain and edit brain tissue efficiently. At the completion of this proposed study, we will have developed an efficient strategy for gene editing neurons using a novel non-viral delivery vehicle CRISPR-PEG. Our proposed studies are significant because the results will provide the basis for developing therapeutics for FXS and fragile X-associated disorders caused by FMR1 deficiency. Moreover, we will develop a non-viral-based vehicle that can edit large volumes of brain tissue after a single injection. The experiments in this proposal are highly innovative because we will have developed an efficient and safe non-viral delivery vehicle, which will greatly advance the field of neuroscience and CRISPR-based therapeutics.