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.

Extrachromosomal circular DNA (eccDNA) facilitates adaptive evolution by allowing rapid and extensive gene copy number variation and is implicated in the pathology of cancer and ageing. Here, we demonstrate that yeast aged under environmental copper accumulate high levels of eccDNA containing the copper-resistance gene CUP1. Transcription of the tandemly repeated CUP1 gene causes CUP1 eccDNA accumulation, which occurs in the absence of phenotypic selection. We have developed a sensitive and quantitative eccDNA sequencing pipeline that reveals CUP1 eccDNA accumulation on copper exposure to be exquisitely site specific, with no other detectable changes across the eccDNA complement. eccDNA forms de novo from the CUP1 locus through processing of DNA double-strand breaks (DSBs) by Sae2, Mre11 and Mus81, and genome-wide analyses show that other protein coding eccDNA species in aged yeast share a similar biogenesis pathway. Although abundant, we find that CUP1 eccDNA does not replicate efficiently, and high-copy numbers in aged cells arise through frequent formation events combined with asymmetric DNA segregation. The transcriptional stimulation of CUP1 eccDNA formation shows that age-linked genetic change varies with transcription pattern, resulting in gene copy number profiles tailored by environment.

Extrachromosomal circular DNA (eccDNA) facilitates adaptive evolution by allowing rapid and extensive gene copy number variation, and is implicated in the pathology of cancer and ageing. Here, we demonstrate that yeast aged under environmental copper accumulate high levels of eccDNA containing the copper resistance gene CUP1. Transcription of CUP1 causes CUP1 eccDNA accumulation, which occurs in the absence of phenotypic selection. We have developed a sensitive and quantitative eccDNA sequencing pipeline that reveals CUP1 eccDNA accumulation on copper exposure to be exquisitely site specific, with no other detectable changes across the eccDNA complement. eccDNA forms de novo from the CUP1 locus through processing of DNA double-strand breaks (DSBs) by Sae2 / Mre11 and Mus81, and genome-wide analyses show that other protein coding eccDNA species in aged yeast share a similar biogenesis pathway. Although abundant we find that CUP1 eccDNA does not replicate efficiently, and high copy numbers in aged cells arise through frequent formation events combined with asymmetric DNA segregation. The transcriptional stimulation of CUP1 eccDNA formation shows that age-linked genetic change varies with transcription pattern, resulting in gene copy number profiles tailored by environment.

DNA double-strand breaks are repaired by multiple pathways, including non-homologous end-joining (NHEJ) and microhomology-mediated end-joining (MMEJ). The balance of these pathways is dependent on the local chromatin context, but the underlying mechanisms are poorly understood. By combining knockout screening with a dual MMEJ:NHEJ reporter inserted in 19 different chromatin environments, we identified dozens of DNA repair proteins that modulate pathway balance dependent on the local chromatin state. Proteins that favor NHEJ mostly synergize with euchromatin, while proteins that favor MMEJ generally synergize with distinct types of heterochromatin. BRCA2 is an example of the former, which is corroborated by chromatin-dependent shifts in mutation patterns of BRCA2-/- cancer genomes. These results uncover a complex network of proteins that regulate MMEJ:NHEJ balance in a chromatin context-dependent manner.

Cells respond to double-strand breaks (DSBs) by activating DNA damage response pathways, including cell cycle arrest. We have previously shown that a single double-strand break generated via CRISPR/Cas9 is sufficient to delay cell cycle progression and compromise cell viability. However, we also found that the cellular response to DSBs can vary, independent of the number of lesions. This implies that not all DSBs are equally toxic, and raises the question if the location of a single double-strand break could influence its toxicity. To systematically investigate if DSB-location is a determinant of toxicity we performed a CRISPR/Cas9 screen targeting 6237 single sites in the human genome. Next, we developed a data-driven framework to design CRISPR/Cas9 sgRNA (crRNA) pools targeting specific chromatin features. The chromatin context was defined using ChromHMM states, Lamin-B1 DAM-iD, DNAseI hypersensitivity, and RNA-sequencing data. We computationally designed 6 distinct crRNA pools, each containing 10 crRNAs targeting the same chromatin state. We show that the toxicity of a DSB is highly similar across the different ChromHMM states. Rather, we find that the major determinants of toxicity of a sgRNA are cutting efficiency and off-target effects. Thus, chromatin features have little to no effect on the toxicity of a single CRISPR/Cas9-induced DSB.

17DNA double-strand break (DSB) repair is mediated by multiple pathways, including classical non-18 homologous end-joining pathway (NHEJ) and several homology-driven repair pathways. This is 19 particularly important for Cas9-mediated genome editing, where the outcome critically depends on the 20 pathway that repairs the break. It is thought that the local chromatin context affects the pathway choice, but 21 the underlying principles are poorly understood. Using a newly developed multiplexed reporter assay in 22 combination with Cas9 cutting, we systematically measured the relative activities of three DSB repair 23 pathways as function of chromatin context in >1,000 genomic locations. This revealed that NHEJ is broadly 24 biased towards euchromatin, while microhomology-mediated end-joining (MMEJ) is more efficient in 25 specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 26 methyltransferase EZH2 shifts the balance towards NHEJ. Single-strand templated repair (SSTR), often 27 used for precise CRISPR editing, competes with MMEJ, and this competition is weakly associated with 28 chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway 29 balance, and guidance for the design of Cas9-mediated genome editing experiments. 31 61Much less is known about the impact of chromatin on MMEJ and SSTR. Like HR, these pathways 62 require resection of the DNA ends to produce single-stranded DNA overhangs, but downstream of this step 63 the mechanisms and responsible proteins diverge (Chang et al., 2017; Scully et al., 2019; Yeh et al., 2019). 3It is thus possible that the local chromatin environment also modulates MMEJ and SSTR in unique ways, 65 but this has remained largely unexplored (Clouaire and Legube, 2019; Mitrentsi et al., 2020). 66One strategy to investigate the impact of local chromatin context on repair pathway balance is to 67 generate DSBs at various genomic locations with known chromatin states, and compare pathway utilization 68 across these locations (van Overbeek et al., 2016; Clouaire et al., 2018; Chakrabarti et al., 2019). However, 69 with such an approach it is difficult to separate the effects of chromatin context from the effects of sequence 70 context, because both vary simultaneously along the genome. Ideally, different chromatin contexts are 71 compared while the sequence context is kept fixed. 72Here, we report a strategy that effectively tackles these challenges in human cells. The strategy 73 consists of two parts. First, we developed a reporter that, when cut with Cas9, produces distinct "scars" 74 when repaired by either NHEJ, MMEJ or SSTR; high-throughput sequencing of these scars provides highly 75 accurate measurements of the relative activities of the three pathways. Second, we used a modification of 76 our TRIP method (Akhtar et al., 2013) to insert this reporter into >1,000 random genomic locations, tracking 77 each individual reporter in parallel by molecular barcoding. We thus systematically measured the r...

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The efficiency and outcome of CRISPR/Cas9 editing depends on the chromatin state at the cut site. It has been shown that changing the chromatin state can influence both the efficiency and repair outcome, and epigenetic drugs have been used to improve Cas9 editing. However, because the target proteins of these drugs are not homogeneously distributed across the genome, the efficacy of these drugs may be expected to vary from locus to locus. Here, we systematically analyzed this chromatin context-dependency for 160 epigenetic drugs. We used a human cell line with 19 stably integrated reporters to induce a double-stranded break (DSB) in different chromatin environments. We then measure Cas9 editing efficiency and repair pathway usage by sequencing the mutational signatures. We identified 67 drugs that modulate Cas9 editing efficiency and/or repair outcome dependent on the local chromatin environment. For example, we find a subset of histone deacetylase inhibitors that improve Cas9 editing efficiency throughout all types of heterochromatin (e.g., PCI-24781), while others were only effective in H3K27me3-marked regions (e.g., Vorinostat). In summary, this study reveals that most epigenetic drugs alter CRISPR editing in a chromatin-dependent manner, and provides a detailed guide to improve Cas9 editing more selectively at the desired location.