2. Academic Validation
  3. Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance

Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance

  • Mol Cell. 2021 May 20;81(10):2216-2230.e10. doi: 10.1016/j.molcel.2021.03.032.
Ruben Schep 1,  Eva K Brinkman 1,  Christ Leemans 1,  Xabier Vergara 2,  Robin H van der Weide 1,  Ben Morris 3,  Tom van Schaik 1,  Stefano G Manzo 1,  Daniel Peric-Hupkes 1,  Jeroen van den Berg 4,  Roderick L Beijersbergen 3,  René H Medema 4,  Bas van Steensel 5
Affiliations

Affiliations

  • 1 Oncode Institute, Netherlands Cancer Institute, 1066 CX, Amsterdam, the Netherlands; Division of Gene Regulation, Netherlands Cancer Institute, 1066 CX, Amsterdam, the Netherlands.
  • 2 Oncode Institute, Netherlands Cancer Institute, 1066 CX, Amsterdam, the Netherlands; Division of Gene Regulation, Netherlands Cancer Institute, 1066 CX, Amsterdam, the Netherlands; Division of Cell Biology, Netherlands Cancer Institute, 1066 CX, Amsterdam, the Netherlands.
  • 3 Division of Molecular Carcinogenesis, Netherlands Cancer Institute, 1066 CX, Amsterdam, the Netherlands; Robotics Screening Center, Netherlands Cancer Institute, 1066 CX, Amsterdam, the Netherlands.
  • 4 Oncode Institute, Netherlands Cancer Institute, 1066 CX, Amsterdam, the Netherlands; Division of Cell Biology, Netherlands Cancer Institute, 1066 CX, Amsterdam, the Netherlands.
  • 5 Oncode Institute, Netherlands Cancer Institute, 1066 CX, Amsterdam, the Netherlands; Division of Gene Regulation, Netherlands Cancer Institute, 1066 CX, Amsterdam, the Netherlands; Department of Cell Biology, Erasmus University Medical Centre, 3015 CN, Rotterdam, the Netherlands. Electronic address: b.v.steensel@nki.nl.
PMID: 33848455 DOI: 10.1016/j.molcel.2021.03.032
Abstract

DNA double-strand break (DSB) repair is mediated by multiple pathways. It is thought that the local chromatin context affects the pathway choice, but the underlying principles are poorly understood. Using a multiplexed reporter assay in combination with Cas9 cutting, we systematically measure the relative activities of three DSB repair pathways as a function of chromatin context in >1,000 genomic locations. This reveals that non-homologous end-joining (NHEJ) is broadly biased toward euchromatin, while the contribution of microhomology-mediated end-joining (MMEJ) is higher in specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 methyltransferase EZH2 reverts the balance toward NHEJ. Single-stranded template repair (SSTR), often used for precise CRISPR editing, competes with MMEJ and is moderately linked to chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway balance and guidance for the design of Cas9-mediated genome editing experiments.

Keywords

CRISPR; Chromatin; DNA repair; MMEJ; NHEJ; SSTR; double strand break; heterochromatin; nuclear lamina; reporter assay.

Figures
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  • HY-101570
    99.70%, DNA-PK Inhibitor