Nearly half of all mammalian genomes originate from mobile sequences and ancient retroviruses called RetroElements (REs). REs consists of tens-of-thousands of nearly identical repetitive copies per RE-family and have been historically considered “Molecular Parasites” or “Junk DNA” as most are immobilized by mutations and epigenetic silencing. However, rare instances of gene disruption following RE integration have led to an “US versus THEM” model that fails to reflect symbiotic relationships developed over millions of years of co- evolution. This is best demonstrated by the well-known but poorly understood phenomenon of “RE-reactivation” in mammalian preimplantation embryos, in which thousands of related REs express simultaneously at distinct stages. REs are essential for development, as their disruption is embryonic lethal. Some REs have maintained regulatory features that influence expression, structure, and function of nearby genes and RE-signatures tightly overlap with events that are unique to preimplantation embryos, including zygotic genome activation, totipotency, reprogramming and pluripotency, supporting the hypothesis that RE-reactivation was perhaps the innovative catalysts for promoting conserved embryo specific programs through domestication of viral functions and rewired gene networks. In support, our single cell technologies and preimplantation RNA-Seq analysis to assess RE function revealed highly conserved expression patterns of RE families across 8 mammalian species. To study these, we developed an embryo editing technology (CRISPR-EZ) to delete 5 specific RE insertions in mice, each resulting in unique developmental defects. These findings revealed the first essential retrotransposon in mammalian development and established reactivation as part of “Normal Development” and shifting our view on “Junk DNA”. While silenced in most tissues, unintentional RT reactivation is commonly observed (and largely ignored) during epigenetic breakdown occurring in aging, autoimmunity, neurodegeneration, and cancer. Therefore, the goal of this proposal is to characterize RE regulatory networks and mechanisms that ensure proper development and leverage this information to explore the therapeutic potential of “synthetic RE- reactivation” to re-engage beneficial “emergent properties” in epigenetically compromised cells or block RE- Signatures in compromised cells to ameliorate or even reverse pathologies. This proposal pioneers a combination of comparative biology, genome editing and parallel in vivo / in vitro strategies using CRISPR/CAS9 variants and consists of three interconnected but independent aims. We use a human centric criterion to select RE candidates to 1) characterize conserved RE-mechanisms in development through humanized mouse models, 2) define RE regulation through a combined in vitro/in vivo CRISPR strategy and 3) assess the therapeutic potential of RE modulation in adult and diseased cells. Together, our findings will provide an...