CRISPR/Cas-9 is an adaptive immune system which has been repurposed as a gene editing technology in living cells. Genome editing relies on the repair of Cas9-induced, site-specific DNA double-strand breaks (DSBs), which are the most deleterious type of DNA damage in cells that can prevent the progression of replication and the transcription of important genes. For DSBs to be effectively repaired, it goes through Homology Directed Repair (HDR), which is considered an error free mechanism because it utilizes a donor DNA template strand to provide precise changes to the DNA. While HDR, being highly competent to inserting or correcting sequences, is desirable in the context of gene editing applications, its efficiency is limited by other competing, higher frequency, error-prone pathways such as Non-homologous End Joining (NHEJ) and Microhomology-Mediated End Joining (MMEJ), both of which produce undesirable outcomes during gene editing. Thus, competition with end-joining factors by NHEJ and MMEJ pose major barriers that need to be overcome to ensure efficient, precise genome editing by HDR.<br/>Previous work from this lab has identified an engineered variant of RAD18, e18, as a potent stimulator in inducing CRISPR-mediated HDR by inhibiting 53BP1, an important NHEJ promoting factor, from localizing to the break site. We then wanted to know whether stimulation of HDR was coming at the expense of MMEJ since little is known about the effects of e18 on this repair pathway. Preliminary data indicates that in e18-expressing cells, there is a decrease in NHEJ compared to the control at the cleave site of Cas-9 which leads us to believe that NHEJ is being inhibited as e18 promotes HDR-dependent precise gene editing. We are aiming to validate whether e18 can be a potential inhibitor of MMEJ to boost HDR at multiple loci in various human cells. To determine e18’s impact on repair outcomes, we needed to analyze the frequency of mutations and all the repair products introduced at the DNA break site using Next Generation Sequencing (NGS). Overall, the functional data can potentially reveal that this novel genetically encoded enhancer of CRISPR-mediated HDR is inhibiting or decreasing MMEJ pathways and lead to the development of improved CRISPR-based technologies and disease therapies.<br/>

Effect of over-expression of an engineered RAD18 variant on repair outcomes at CRISPR/Cas9 induced double stranded breaks

2019 Spring National Meeting

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