Development of tools for rapid systematic refinement of in vivo gene editing technologies

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Abstract Genome sequencing efforts are increasingly revealing gene variants that disrupt tissue development and function. Therapies for genetic disorders are currently limited by our inability to make precise and permanent adjustments to dysfunctional genes and associated regulatory programs. However, CRISPR/Cas9- based genome editing is proving to be a powerful gene regulatory tool with tremendous therapeutic potential. One particularly promising approach is the use of adeno-associated virus (AAV) to deliver CRISPR/Cas9 components as well as a template for homology directed repair (HDR; AAV-HDR). In vitro AAV-HDR efficiency can be spectacularly high, with >90% of transduced cells correctly edited in some cases, while in vivo studies have demonstrated more modest, and highly variable, results. To successfully employ AAV-HDR in a therapeutic setting, its efficiency will need to be optimized. In addition, a robust understanding of AAV-HDR mechanisms will be necessary to ensure safety. Unfortunately, efforts to study and improve AAV-HDR have been severely hampered by a lack of tools that allow for high-throughput, systematic analyses. Hypothesis: Development of high-throughput methodologies for measuring in vivo AAV-HDR editing efficiency will enable rapid discovery of the underlying molecular mechanisms and facilitate optimization necessary for clinical translation. This proposal will develop and deploy the tools necessary for rapid, systematic refinement of in vivo AAV-HDR. In Aim 1, using mice as a model organism, we will develop a method for simultaneously measuring AAV-HDR efficiency at many target loci. We will investigate the locus-dependent variability of AAV-HDR efficiency by utilizing the system to analyze the relationship between efficiency and target locus chromatin state in cardiomyocytes. In Aim 2, we will develop a high-throughput method, based on a pooled CRISPR- knockout screen, for assessing the impact of gene perturbations on cardiac AAV-HDR efficiency. We will use the system to gain insights into the molecular mechanism of AAV-HDR, by identifying DNA-repair factors that are necessary for successful gene editing. AAV-HDR can occur at high efficiency within heart muscle cells, although efficiency varies dramatically by target locus. Here we propose development of two systems that will leverage next-generation sequencing to make many parallel measurements of AAV-HDR efficiency. To our knowledge, both systems will be the first of their kind. Our use of these systems will lead to key conceptual advances in understanding the mechanisms underlying AAV-HDR. We anticipate that these technical and conceptual advances will promote development of AAV-HDR based therapies.