Double-strand break toxicity is chromatin context independent.

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.

From fluorescent foci to sequence: Illuminating DNA double strand break repair by high-throughput sequencing technologies.

Technologies to study DNA double-strand break (DSB) repair have traditionally mostly relied on fluorescence read-outs, either by microscopy or flow cytometry. The advent of high throughput sequencing (HTS) has created fundamentally new opportunities to study the mechanisms underlying DSB repair. Here, we review the suite of HTS-based assays that are used to study three different aspects of DNA repair: detection of broken ends, protein recruitment and pathway usage. We highlight new opportunities that HTS technology offers towards a better understanding of the DSB repair process.

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 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.

Transcription-induced formation of extrachromosomal DNA during yeast ageing.

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.