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

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 re...

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Published in:Molecular cell Vol. 81; no. 10; p. 2216
Main Authors: Schep, Ruben, Brinkman, Eva K, Leemans, Christ, Vergara, Xabier, van der Weide, Robin H, Morris, Ben, van Schaik, Tom, Manzo, Stefano G, Peric-Hupkes, Daniel, van den Berg, Jeroen, Beijersbergen, Roderick L, Medema, René H, van Steensel, Bas
Format: Journal Article
Language:English
Published: United States 20.05.2021
Subjects:
Base Sequence
Chromatin - metabolism
CRISPR-Associated Protein 9 - metabolism
DNA Breaks, Double-Stranded
DNA End-Joining Repair
DNA Repair
Euchromatin - metabolism
Gene Rearrangement
Genome, Human
Heterochromatin - metabolism
Humans
INDEL Mutation - genetics
K562 Cells
Kinetics
Protein Binding
Reproducibility of Results
CRISPR
MMEJ
Chromatin
double strand break
SSTR
reporter assay
nuclear lamina
NHEJ
DNA repair
heterochromatin
ISSN:1097-4164, 1097-4164
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Summary: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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ISSN:1097-4164
1097-4164
DOI:10.1016/j.molcel.2021.03.032