Establishing a consensus model for the role of genomic context in CRISPR-directed gene editing
Date
2022
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Publisher
University of Delaware
Abstract
CRISPR-mediated gene editing systems have grown rapidly throughout all biological disciplines, due to their ability to facilitate robust gene editing and its ease of use and design. As more research was performed it quickly became apparent that many of the so-called “low-hanging fruit” of clinical gene editing targets, such as sickle cell anemia, would be much more difficult to address than previously believed. Because CRISPR/Cas gene editing systems rely on the DNA repair activity of the cell to introduce genetic changes, variations in activity and interplay among the three main DNA repair pathways (NHEJ, MMEJ, and HDR) lead to variation and on-target disruptions when attempting to target genes for clinically-relevant disease targets. Previously, These complex repair outcomes were not deeply interrogated, due to the high effort and comparatively low efficiency of pre-CRISPR gene editing tools. However, with the increasing applications of CRISPR-based gene editing research, these complex repair outcomes are being seen in more spaces, and at rates that warrant a deeper look and elucidation. In the work presented in this dissertation, I examined the mechanisms of DNA repair in order to determine the role individual gene sequence and genetic context plays in CRISPR/Cas-mediated gene repair. To accomplish this, I developed a new analysis software that allowed for precise interrogation of complex CRISPR editing reactions, called DECODR. Utilizing DECODR, I could rapidly analyze and iterate on the findings of the gene editing outcomes in both in vitro and live-cell gene editing reactions. I proposed a model for how CRISPR/Cas12a can influence editing outcomes and indel directionality, called traumatic dissociation, and synthesized it with a model that I had previously described, called the ExACT model, to create a new, highly comprehensive model for DNA repair that offers much deeper insight into the complex mechanisms that can influence gene editing reacitons across a wide variety of organisms and gene targets.
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Keywords
CRISPR, DECODR, DNA damage, DNA repair, Gene editing