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

doi:10.1016/j.molcel.2021.03.032

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

Cell biology

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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 language	English
Pages (from-to)	2216-2230.e10
Journal	Molecular Cell
Volume	81
Issue number	10
DOIs	https://doi.org/10.1016/j.molcel.2021.03.032
Publication status	Published - 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 .

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© 2021 The Authors

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Impact of chromatin context on Cas9-induced DNAFinal published version, 7.31 MBLicence: CC BY-NC-ND

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Schep, R., Brinkman, E. K., Leemans, C., Vergara, X., van der Weide, R. H., Morris, B., van Schaik, T., Manzo, S. G., Peric-Hupkes, D., van den Berg, J., Beijersbergen, R. L., Medema, R. H., & van Steensel, B. (2021). Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance. Molecular Cell, 81(10), 2216-2230.e10. https://doi.org/10.1016/j.molcel.2021.03.032

@article{3c8c6c4d2eed4b5382cf6d294ed3008e,

title = "Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance",

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.",

author = "Ruben Schep and Brinkman, {Eva K.} and Christ Leemans and Xabier Vergara and {van der Weide}, {Robin H.} and Ben Morris and {van Schaik}, Tom and Manzo, {Stefano G.} and Daniel Peric-Hupkes and {van den Berg}, Jeroen and Beijersbergen, {Roderick L.} and Medema, {Ren{\'e} H.} and {van Steensel}, Bas",

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: {\textcopyright} 2021 The Authors",

year = "2021",

month = may,

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doi = "10.1016/j.molcel.2021.03.032",

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Schep, R, Brinkman, EK, Leemans, C, Vergara, X, van der Weide, RH, Morris, B, van Schaik, T, Manzo, SG, Peric-Hupkes, D, van den Berg, J, Beijersbergen, RL, Medema, RH & van Steensel, B 2021, 'Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance', Molecular Cell, vol. 81, no. 10, pp. 2216-2230.e10. https://doi.org/10.1016/j.molcel.2021.03.032

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AU - Schep, Ruben

AU - Brinkman, Eva K.

AU - Leemans, Christ

AU - Vergara, Xabier

AU - van der Weide, Robin H.

AU - Morris, Ben

AU - van Schaik, Tom

AU - Manzo, Stefano G.

AU - Peric-Hupkes, Daniel

AU - van den Berg, Jeroen

AU - Beijersbergen, Roderick L.

AU - Medema, René H.

AU - van Steensel, Bas

N1 - 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

PY - 2021/5/20

Y1 - 2021/5/20

N2 - 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.

AB - 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.

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JO - Molecular Cell

JF - Molecular Cell

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ER -

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

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