Project Summary/Abstract Combination antiretroviral therapy (cART) is effective at reducing viral load and suppressing HIV-1 infection, however, there still is no cure for HIV-1 disease. This is due in part to the formation of latently infected cells harboring integrated proviruses in tissue and cell reservoirs. Clustered regularly interspaced palindromic repeats (CRISPR) gene editing has shown promise as an HIV cure strategy. All Cas enzymes in use today initiate binding by recognizing a protospacer adjacent motif (PAM) followed by the complementarity between guide RNA (gRNA) and target DNA to induce DNA cleavage. Subsequent double-strand break repair by endogenous cellular processes has been shown to result in a non-random mutational distribution dictated by protospacer and flanking sequence context. Furthermore, natural genetic variation within integrated proviral sequences has been shown to decrease the CRISPR-mediated editing efficiency which is critically dependent for efficacy of the gRNA selection process. The past funding period, we have designed a patented computational pipeline to select broad- spectrum spCas9 gRNAs that account for HIV sequence variation within and between large numbers of individuals and that have no off-target effect using predictive algorithms or functional assays. Preliminary data presented here shows gRNAs have efficacy in other tissue compartments (brain) and across subtypes. In addition to these gRNA design advances, the team also showed major advancements in delivery and effectiveness in small animal studies and non-human primate studies. In order to better harness the utility of CRISPR/Cas gene editing, this project will utilize novel high-throughput biologic assays combined with state-of- the art computational biology to expand what is known about how novel Cas enzymes edit the DNA target in vitro and test HIV-1-infected patient samples ex vivo and in vivo to optimize treatment strategy (Cas:gRNA combination) selection to account for HIV sequence variation within and across tissue compartments (periphery vs brain) and subtypes. Novel resources like the Multiple Lentiviral Expression System (MuLE) and the Mammalian Synthetic Cellular Recorder Integrating Biological Events (mSCRIBE) will be leveraged to study Cas enzymology and HIV-1 reactivation at the single-cell level. We hypothesize that, Cas:gRNA targeting will induce safe and reproducible editing outcomes that are predictablly based on the enzyme, target, and surrounding nucleotide sequence. To interrogate this hypothesis, three Specific Aims will be used: (i) develop a generic model of Cas:gRNA combination repair outcomes, (ii) identify functional impact of Cas:gRNA pairs using molecular recorders, and (iii) ex vivo and in vivo testing of combined Cas:gRNA pairs on HIV. These Aims will extend the knowledge of CRIPSR editing technologies for all fields of biology using the HIV platform. It will do this in cells important for HIV research in the periphery...