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

作者：Ruben Schep, Eva K. Brinkman, Christ Leemans, Xabier Vergara, Robin H. van der Weide, Ben Morris, Tom van Schaik, Stefano Giustino Manzo, Daniel Peric‐Hupkes, Jeroen van den Berg, Roderick L. Beijersbergen, René H. Medema, Bas van Steensel · 发表于：Molecular Cell · 年份：2021 · DOI：10.1016/j.molcel.2021.03.032 · 被引用次数：216 · 研究领域：DNA Repair Mechanisms、CRISPR and Genetic Engineering、Genomics and Chromatin Dynamics

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.