Abstract
Homologous recombination (HR) faithfully restores DNA double-strand breaks. Defects in this HR repair pathway are associated with cancer predisposition. In genetic engineering, HR has been used extensively to study gene function and it represents an ideal method of gene therapy for single gene disorders. Here, we present a novel assay to measure HR in living cells. The HR substrate consisted of a non-fluorescent 3’ truncated form of the eGFP gene and was integrated into the AAVS1 locus, known as a safe harbor. The donor DNA template comprised a 5’ truncated eGFP copy and was delivered via AAV particles. HR mediated repair restored full-length eGFP coding sequence, resulting in eGFP+ cells. The utility of our assay in quantifying HR events was validated by exploring the impact of the overexpression of HR promoters and the siRNA-mediated silencing of genes known to play a role in DNA repair on the frequency of HR. We conclude that this novel assay represents a useful tool to further investigate the mechanisms that control HR and test continually emerging tools for HR-mediated genome editing.
Highlights
DNA double-strand breaks (DSBs) are among the most serious types of DNA damage in cells and can lead to genetic instability and tumorigenesis [1]
homologous recombination assay (HR) leads to the restoration of the full-length enhanced green fluorescent protein (eGFP) coding sequence from two different truncated eGFP copies. eGFP is a variant of the wild-type GFP with higher-intensity emission than GFP [20], facilitating the detection of HR events
We optimized the assay system in the human colon cancer cell line HCT116, which is highly proficient in gene targeting [22, 23] and has been widely used to study the molecular mechanisms of HR [24,25,26]
Summary
DNA double-strand breaks (DSBs) are among the most serious types of DNA damage in cells and can lead to genetic instability and tumorigenesis [1]. DSB may be induced by exogenous genotoxic insults, such as ionizing radiation, and occur spontaneously in various cellular processes including DNA replication and V(D)J recombination. There are two major pathways for DSB repair: non-homologous end joining (NHEJ) and homologous recombination. NHEJ directly ligates the two broken ends of a DSB and is accessible throughout the cell cycle. HR is an error-free repair mechanism that primarily uses the intact sister chromatid as a template for repair and predominates in S-phase cells [2]
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