In partial fulfillment of the requirements for the degree of

Master of Science in Biology

in the

School of Biological Sciences

Yiqi Zhang

Will defend her thesis

Experiments to Study DNA Double-Strand Break Repair by RNA in Human Cells

18 April 2024

3:00 PM

https://gatech.zoom.us/j/96068084892?pwd=d2YxZmFQc1cya1N4NmtIWDhFZVZlZz09

Thesis Advisor:

Dr. Francesca Storici

School of Biological Sciences

Georgia Institute of Technology

Committee Members:

Dr. Yuhong Fan

School of Biological Sciences

Georgia Institute of Technology

 Dr. Shuyi Nie

School of Biological Sciences

Georgia Institute of Technology

Dr. Yonggang Ke

Wallace H. Coulter Department of Biomedical Engineering

Emory University

Abstract: DNA double-strand breaks (DSBs) pose a significant threat to genetic stability. Efforts to address this issue typically fall into two main approaches: preventing the formation of DSBs and enhancing their repair. Given the multitude of factors that can cause DSBs, directly inhibiting their occurrence at the source presents challenges. Consequently, research has primarily focused on understanding and harnessing DSB repair mechanisms, leading to the identification of three primary pathways: homologous recombination (HR), non-homologous end joining (NHEJ), and microhomology-mediated end joining (MMEJ). These mechanisms rely on DNA as a repair template, yet recent studies have highlighted the potential role of RNA, which is abundant in cells and complements the transcription template DNA, as an alternative repair template. Previous findings suggest that RNA not only directly participates in DNA repair in yeast cells, but also facilitates repair at exon-intron junctions in a sequence-dependent manner in human cells. Several unresolved questions stemming from these investigations include the impact of intronic sequences on RNA facilitation, the influence of RNA on DSB repair in exons, and the effect of the size of DNA gaps induced by two DSBs on repair processes. In this study, we took advantage of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system with the CRISPR Associated Protein 9 (Cas9) and the single-guide RNAs (sgRNAs) for generating specific DSBs and study the direct role of RNA in DSB repair in human cells. We designed three sgRNAs for single-DSB cleavage systems and two sets of sgRNAs for double-DSB cleavage systems targeting the first exon of the DsRed marker gene on a plasmid. To evaluate the efficiency of the single and double-DSB cleavage systems, an in vitro cleavage assay was conducted, and the cleavage efficiency of each sgRNA and sgRNA set was assessed. Based on these results, sgRNAs E, I, J, as well as sgRNA pairs E+I and E+J, were selected for further in vivo experiments in the human embryonic kidney cells expressing the T antigen for replication of the plasmid (HEK293T cells). Subsequently, repair frequencies in each genetic construct within the DsRed exon were examined in vivo using HEK293T wild-type cells transfected with plasmids expressing the CRISPR cleavage systems. Analysis of next-generation sequencing (NGS) data from the HEK293T sequencing libraries allowed for the differentiation of repair products based on their characteristics. Additionally, the frequencies of MMEJ and NHEJ repair following a single DSB or DNA gaps induced by the cleavage systems in each construct were calculated. Our results indicate a clear distinction: unlike DSBs occurring at exon-intron junctions, the presence of an RNA transcript does not affect the efficiency of gap deletion, irrespective of splicing, when DSBs are within the same exon sequence of the DsRed gene. Moreover, when the transcription level of the RNA is low, the DSB repair outcomes are mainly the same as when the transcription level is high.