Developing an in vitro system to elucidate the mechanism and regulation of CRISPR-directed gene editing and the responding DNA damage repair pathways
Date
2020
Authors
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Publisher
University of Delaware
Abstract
Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)-directed gene editing is a revolutionary approach to genetic manipulation and disease modeling. CRISPR-Cas systems have demonstrated utility for a wide-range of applications and biotechnological breakthroughs. Yet, challenges remain including the unpredictability of genetic outcomes generated as a result of CRISPR-directed DNA cleavage and unbalanced responses by DNA damage repair pathways. Site-specific DNA breakage introduced by Cas proteins is followed by a DNA damage repair response from one of two competing pathways, Non-Homologous End Joining (NHEJ) or Homology Directed Repair (HDR). Therefore, the influence of CRISPR-directed DNA damage as a regulating factor of these response pathways should provide a key to understanding this unpredictability. We propose to address this gap in knowledge by developing an in vitro gene editing system to study the factors affecting the mechanism and regulation of DNA repair in response to CRISPR-directed DNA disruption. The ability to assess CRISPR-Cas site-specific DNA disruption and damage responses in a simplified system will facilitate a deeper understanding of the capabilities and limitations of gene editing tools. The ability to initiate CRISPR-directed gene editing reactions with a methodical regulatory approach of strategically incorporated gene editing tools will provide a foundational system for identifying the regulatory factors affecting precise gene editing event. This will provide insight and a deeper understanding of the regulatory factors involved in CRISPR-directed gene editing and the diversity of genetic outcomes generated by competing DNA repair responses. We have been able to generate in vitro models for several unique gene editing reactions. This system enables us to gain new insights into the mechanisms of repair while providing the opportunity to assess the diversity of genetic outcomes resulting from gene editing reactions. This in vitro gene editing system will continue to provide new insight into the regulatory mechanisms of CRISPR gene editing technologies, many of which remain to be fully understood and defined.
Description
Keywords
Cas12a, Cas9, CRISPR, CRISPR-Cas, DNA repair, Gene editing