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

Abstract

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.

The Sound of Silence: How Silenced Chromatin Orchestrates the Repair of Double-Strand Breaks

“…In contrast, a recent study that used a sequencing-based reporter screen to investigate the impact of chromatin context on CRISPR-Cas9-induced DSBs did reveal differences in repair pathway usage in H3K27me3 regions [ 127 ]. This reporter was randomly integrated in >1000 different locations in the genome of human cancer cells and can be cleaved by Cas9.…”

Tissue Specific DNA Repair Outcomes Shape the Landscape of Genome Editing

“…NHEJ, on the other hand, is preferred in heterochromatic regions and at sites where H4 is demethylated at lysine 20 (H4K20me2; Karakaidos et al, 2020). Recently, the pathway balance between NHEJ and MMEJ as influenced by chromatin configuration has also been mapped (Schep et al, 2021). This study showed that MMEJ is more active than NHEJ in specific heterochromatin contexts, namely late replicating regions, lamina associated regions, and at H3K9me2 sites.…”

“…This study showed that MMEJ is more active than NHEJ in specific heterochromatin contexts, namely late replicating regions, lamina associated regions, and at H3K9me2 sites. Moreover, MMEJ was shown to compete with SSTR (Schep et al, 2021). Therefore, systematically mapping chromatin environments across cell types can inform avenues for regulation to successfully install CRISPR edits which rely on the incorporation of repair templates.…”

DNA Double-Strand Break Repair: All Roads Lead to HeterochROMAtin Marks

“…In addition to H3K9me3, H3K27me3 also decorates heterochromatin regions, such as those associated with the lamina, and DSBs in these H3K27me3-enriched regions show increased repair by MMEJ ( Lemaître et al, 2014 ; Schep et al, 2021 ). Interestingly, chemical inhibition of H3K27 and not of H3K9 methyltransferases shifted the MMEJ/NHEJ balance toward NHEJ ( Schep et al, 2021 ), arguing that the H3K27me3 heterochromatin mark either stimulates MMEJ or inhibits NHEJ. However, the underlying molecular mechanisms still need to be elucidated and the impact of H3K27me3 on HR is unknown.…”