RNA-mediated DNA double-strand break repair in mammalian cells
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Jeon, Youngkyu
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Abstract
A double-strand break (DSB) is one of the most dangerous legions threatening genome stability, which could cause mutations, genome rearrangement and cell death. We have demonstrated that RNA is an alternative template for the repair of DSB in DNA. In addition to observing RNA-templated DNA repair by synthetic RNA oligonucleotides in yeast and human cells, we showed that endogenous transcript RNA can directly template DSB repair in its own DNA in cis in yeast cells. Here, we examined whether RNA can also mediate DNA DSB repair in mammalian cells using its complementary sequence to DNA. We developed a genetic assay to study RNA-mediated DNA DSB repair in mammalian cells by transferring the yeast ‘cis system’ into a DNA plasmid (cis plasmid) to perform a transient assay. We exploit the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system with the Cas9 endonuclease and guide RNA (gRNA) to direct Cas9 to the chosen target site on the cis plasmid to generate a DSB. Products of DNA DSB repair on the cis plasmid were captured by Next-Generation Sequencing (NGS) by our customized NGS library prep method, which enables sequencing near the DSB site of the cis plasmid. We induced a DSB (1 DSB) or a DNA gap (2 DSBs) to study how a transcript RNA impacts DNA DSB repair in a sequence-dependent manner in human cells through specific DSB repair mechanisms such as non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and RNA-templated DSB repair (R-TDR). Frequencies of DNA DSB repair were determined by the number of sequencing reads, and specific sequences produced by each DSB repair mechanisms were used to estimate frequencies of each DSB repair mechanisms. Through the NGS data analyses, this work uncovers that endogenous RNA transcripts can mediate DNA DSB repair in a sequence-dependent manner through NHEJ, MMEJ, and R-TDR. For example, we find that the presence or absence of an intron in the RNA sequence of a transcribed gene that experiences a DSB markedly impacts DSB repair by MMEJ. Moreover, we show that a transcript RNA promotes repair of a gap in its own DNA gene by NHEJ. Our results reveal a profound role of RNA is shaping the way DSBs are repaired in DNA of human cells and provides new avenues for applications in genome editing.
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Date
2022-04-14
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Dissertation