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

Ruben Schep, Eva K. Brinkman, Christ Leemans, Xabier Vergara, Robin H. van der Weide, Ben Morris, Tom van Schaik, Stefano G. Manzo, Daniel Peric-Hupkes, Jeroen van den Berg, Roderick L. Beijersbergen, René H. Medema, Bas van Steensel*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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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.

Original languageEnglish
Pages (from-to)2216-2230.e10
JournalMolecular Cell
Volume81
Issue number10
DOIs
Publication statusPublished - 20 May 2021

Bibliographical note

Funding Information:
We thank the NKI Genomics, Flow Cytometry, and Research High Performance Computing core facilities for excellent support and members from our laboratories for inspiring and helpful discussions. We thank Luca Braccioli and the NKI Protein core facility for Tn5 protein. The NKI Protein Facility is an Instruct-ERIC center. This work was supported by ZonMW TOP grant 91215067 (to R.H.M. and B.v.S.), European Research Council (ERC) Advanced Grant 694466 (to B.v.S), and NIH Common Fund “4D Nucleome” Program grant U54DK107965 (B.v.S.). S.G.M. is funded by Marie Curie /AIRC iCARE2.0 fellowship 800924. The Oncode Institute is partly supported by KWF Dutch Cancer Society .

Publisher Copyright:
© 2021 The Authors

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