Optimizing Cas9-mediated HDR in HEK293

Abstract

The most widely used genome editing tool is the Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated proteins (Cas) system. With its programmable guide RNA sequence for specific gene targeting, it allows for precise genome editing including substitutions, knock-outs, and knock-ins. For the treatment of genetic disorders, a precise edit is compulsory compared to inaccurate end-joining modifications. Our focus will be on Homology Directed Repair (HDR) pathway as it is more precise but is limited by its efficiency and dominance of non-homologous end joining (NHEJ). It has been identified that the proximity of repair template is a rate limiting factor of HDR. Since previous studies has shown that the “all-in-one” method with ssODNs has limitations in replacing large fragments, we will use plasmid donors to counter this limitation. This project aims to improve HDR efficiency, by increasing proximity of ideal donor repair template to repair site via fusing donor plasmid to gRNA. Our findings show that plasmid donors are able to achieve a consistent HDR efficiency.