The Chromatin Structure of CRISPR-Cas9 Target DNA Controls the Balance between Mutagenic and Homology-Directed Gene-Editing Events

Josephine M Janssen; Xiaoyu Chen; Jin Liu; Manuel A F V Gonçalves

doi:10.1016/j.omtn.2019.02.009

The Chromatin Structure of CRISPR-Cas9 Target DNA Controls the Balance between Mutagenic and Homology-Directed Gene-Editing Events

Mol Ther Nucleic Acids. 2019 Jun 7:16:141-154. doi: 10.1016/j.omtn.2019.02.009. Epub 2019 Feb 20.

Authors

Josephine M Janssen¹, Xiaoyu Chen¹, Jin Liu¹, Manuel A F V Gonçalves²

Affiliations

¹ Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands.
² Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands. Electronic address: m.f.v.goncalves@lumc.nl.

Abstract

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 Krüppel-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.

Keywords: CRISPR-Cas9 nucleases; DNA repair pathways; donor DNA; double-stranded DNA breaks; euchromatin; gene-editing outcomes; heterochromatin; homologous recombination; non-homologous end joining.