Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing

Abstract

We present a genome-wide method to map DNA double-strand breaks (DSBs) at nucleotide resolution by direct in situ breaks labeling, enrichment on streptavidin, and next-generation sequencing (BLESS). We comprehensively validated and tested BLESS using different human and mouse cells, DSBs-inducing agents, and sequencing platforms. BLESS was able to detect telomere ends, Sce endonuclease-induced DSBs, and complex genome-wide DSBs landscapes. As a proof of principle, we characterized the genomic landscape of sensitivity to replication stress in human cells, and identified over two thousand non-uniformly distributed aphidicolin-sensitive regions (ASRs) overrepresented in genes and enriched in satellite repeats. ASRs were also enriched in regions rearranged in human cancers, with many cancer-associated genes exhibiting high sensitivity to replication stress. Our method is suitable for genome-wide mapping of DSBs in various cells and experimental conditions with a specificity and resolution unachievable by current techniques.

Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq

“…We chose 10 sgRNAs that had been analyzed individually using GUIDE-seq (Tsai et al 2015), which is likely to be more sensitive than IDLV capture and other methods. Note that BLESS captures a snapshot of DSBs at a given moment of cell fixation (Crosetto et al 2013) and that HTGTS relies on two concurrent DSBs, rather than one, induced in a single cell (Chiarle et al 2011). We digested human genomic DNA with a mixture of the Cas9 protein, the 10 sgRNAs, and one additional sgRNA targeted to the HBB gene, which we had analyzed in our previous study (Kim et al 2015), and carried out two independent WGS analyses ( Fig.…”

“…Both genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq) (Tsai et al 2015) and integration-deficient lentiviral vector (IDLV) capture (Wang et al 2015) rely on NHEJ-mediated insertions of small duplex oligonucleotides or lentiviral vectors, respectively, at cleavage sites. Direct in situ breaks labeling, enrichment on streptavidin, and next-generation sequencing (BLESS) is a method of capturing DSBs in fixed cells (Crosetto et al 2013;Ran et al 2015). Digenome-seq (digested genome sequencing) relies on whole-genome sequencing (WGS) of cell-free genomic DNA digested in vitro using a nuclease of interest (Kim et al 2015).…”

DROSHA associates to DNA damage sites and is required for DNA repair

“…This technology allows the study of the recruitment of a given protein at DNA ends. Briefly, in fixed and permeabilized cells, DNA ends are blunted and ligated to a biotinylated oligonucleotide (Crosetto et al, 2013) which permanently tags DSB-ends; then PLA is performed using primary antibodies against biotin and a partner antibody against the protein of interest, DROSHA in this case (see cartoon in Figure 3E). By this approach, we observed a significant increase of DI-PLA signals upon DNA damage generation, indicating DROSHA presence in close proximity to DNA ends of DSBs ( Figure 3F and G).…”

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

“…Fourth, we excluded sites showing "clustered hot-spots" in which high variability in mutations rates is found within neighboring sequences that represent indels that are likely to arise by technical artifacts and repetitive sequences errors. 48,49 Fifth, indels with no PAM, or with PAM located >10 bp away from seed sequence, or if guide-seed mismatch was more than 5 bps were filtered out as likely technical artifacts 5,15,20,50 (the "seed sequence" has been defined as 10-most proximal nucleotides to PAM). 3,51 Using these criteria, we identified 16 genes in which ICLV-CRISPR/Cas9 induced noticeable changes ( Figure 3C).…”