Journal
MOLECULAR THERAPY-NUCLEIC ACIDS
Volume 16, Issue -, Pages 141-154Publisher
CELL PRESS
DOI: 10.1016/j.omtn.2019.02.009
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Funding
- Dutch Prinses Beatrix Spierfonds [W.OR11-18]
- European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant [765269]
- Duchenne Parent Project NL [17.012]
- ProQR Therapeutics
- China Scholarship Council-Leiden University Joint Scholarship Programme
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Gene editing based on homology-directed repair (HDR) depends on donor DNA templates and programmable nucleases, e.g., RNA-guided CRISPR-Cas9 nucleases. However, next to inducing HDR involving the mending of chromosomal double-stranded breaks (DSBs) with donor DNA substrates, programmable nucleases also yield gene disruptions, triggered by competing non-homologous end joining (NHEJ) pathways. It is, therefore, imperative to identify parameters underlying the relationship between these two outcomes in the context of HDR-based gene editing. Here we implemented quantitative cellular systems, based on epigenetically regulated isogenic target sequences and donor DNA of viral, non-viral, and synthetic origins, to investigate gene-editing outcomes resulting from the interaction between different chromatin conformations and donor DNA structures. We report that, despite a significantly higher prevalence of NHEJ-derived events at euchromatin over Kriippel-associated box (KRAB)-impinged heterochromatin, HDR frequencies are instead generally less impacted by these alternative chromatin conformations. Hence, HDR increases in relation to NHEJ when open euchromatic target sequences acquire a closed heterochromatic state, with donor DNA structures determining, to some extent, the degree of this relative increase in HDR events at heterochromatin. Finally, restricting nuclease activity to HDR-permissive G2 and S phases of the cell cycle through a Cas9-Geminin construct yields lower, hence more favorable, NHEJ to HDR ratios, independently of the chromatin structure.
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