Abstract P2158: In Vivo Dissection Of A CRISPR/Cas9-Mediated Precise Genome Editing Mechanism

Abstract

Genomic sequencing has uncovered many genetic variations that interfere with development and function. Our inability to make precise and permanent genome edits limits our ability to treat genetic disorders. However, CRISPR/Cas9 has been demonstrated to be a potent tool for genome editing, with enormous therapeutic potential. Furthermore, the use of adeno-associated virus (AAV) to deliver CRISPR/Cas9 components, as well as a donor template for homology directed repair (HDR; AAV-HDR), has shown impressive editing efficiencies, particularly in vitro . In comparison, in vivo studies have shown more modest editing efficiencies, with large variation between target loci. Thus, to employ this strategy therapeutically it is imperative to establish a robust understanding of the underlying mechanism, which can then be exploited to improve efficiency. Thus far, the absence of high-throughput tools that enable systematic analysis has hindered efforts to explore and enhance AAV-HDR efficiency. Here we employ two novel high-throughput methodologies for measuring in vivo AAV-HDR editing efficiency in cardiomyocytes of Cas9-expressing mice. In our first approach we analyze the locus-dependent variability of AAV-HDR efficiency, with the goal of identifying the genomic characteristics of high-efficiency loci. In the second approach, we utilize a high-throughput pooled CRISPR KO screen to identify the DNA-repair factors required for efficient gene editing. This approach has uncovered several novel regulators of cardiac AAV-HDR, which we are currently working on characterizing. Collectively these studies will provide insights into the molecular mechanism of AAV-HDR, and we anticipate that these advances will promote development of AAV-HDR based therapies.