Double-stranded break can be repaired by single-stranded oligonucleotides via the ATM/ATR pathway in mammalian cells |
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Authors: | Wang Zai Zhou Zhong-Jun Liu De-Pei Huang Jian-Dong |
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Institution: | Department of Biochemistry, The University of Hong Kong, Hong Kong SAR, People's Republic of China. |
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Abstract: | Single-stranded oligonucleotide (SSO)-mediated gene modification is a newly developed tool for site-specific gene repair in mammalian cells; however, the corrected cells always show G2/M arrest and cannot divide to form colonies. This phenomenon and the unclear mechanism seriously challenge the future application of this technique. In this study, we developed an efficient SSO-mediated DNA repair system based on double-stranded break (DSB) induction. We generated a mutant EGFP gene with insertions of 24 bp to 1.6 kb in length as a reporter integrated in mammalian cell lines. SSOs were successfully used to delete the insertion fragments upon DSB induction at a site near the insertion. We demonstrated that this process is dependent on the ATM/ATR pathway. Importantly, repaired cell clones were viable. Effects of deletion length, SSO length, strand bias, and SSO modification on gene repair frequency were also investigated. |
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