Summary Down syndrome arises from the triplication of a subset of genes on chromosome 21 (HSA21). Individuals with DS uniformly demonstrate some degree of mental retardation (MR). The MR has been attributed to impairments in brain development (i.e. neurogenesis) as well as progressive cell death with altered synaptogenesis (i.e. neurodegeneration). Silencing of one of the three HSA21 chromosomes rescues the DS phenotype but such a therapeutic approach is limited practically by the efficiency of genomic editing and ability to specifically target a single HSA21 chromosome. Our preliminary studies show the feasibility of a novel modified CRISPR approach which greatly enhances the integration of genomic material on HSA21 and we have also devised a means with which to specifically target a single HSA21 copy. We now propose experiments to assess the efficiency of these approaches in human DS iPSC lines and following their differentiation into neural and oligodendrocyte states. We will also address to what extent these interventions normalize both genetic and epigenetic expression within the DS lines compared to their isogenic counterparts and whether the DS cells are rescued from a functional perspective (proliferation, cell death, mitochondrial function and oxidative stress). Overall, if successful, these studies will overcome two fundamental hurdles needed to treat DS (and other chromosomal abnormalities) from an epigenetic approach and lay the foundation for potential animal studies.