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. With prior funding from NIH INCLUDE, we have developed 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 by SNP based PAM targeting with gRNA. Feasibility has been shown in culture model systems. We now propose experiments to assess the efficiency of these approaches in the TcMAC1 mouse model of DS. The TcMAC21 mouse contains 93% of HSA21q protein coding genes that are expressed and regulatable. Overall, if successful, these studies would show the potential ability to reverse, at least partially, the DS neurological phenotype. The funded proposal is the necessary next step to ascertain whether the efficiency of silencing by XIST of a third HSA21 copy in vivo would translate into an observable clinical effect.