Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases

Abstract

Although great progress has been made in the characterization of off-target effects of engineered nucleases, sensitive and unbiased genome-wide methods for the detection of off-target cleavage events and potential collateral damage are still lacking. Here we describe a linear amplification–mediated modification of a previously published high-throughput, genome-wide translocation sequencing (HTGTS) method that robustly detects DNA double-stranded breaks (DSBs) generated by engineered nucleases across the human genome based on their translocation to other endogenous or ectopic DSBs. HTGTS with different Cas9:sgRNA or TALEN-nucleases revealed off-target hotspots for given nucleases that ranged from a few or none to dozens or more, and extended the number of known off-targets for certain previously characterized nucleases by more than 10-fold. We also identified translocations between bona fide nuclease targets on homologous chromosomes, an undesired collateral effect that has not been described. Finally, HTGTS confirmed that the Cas9D10A paired nickase approach suppresses off-target cleavage genome-wide.

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

“…These nucleases, however, can induce off-target mutations, limiting their utility in research and medicine (Cradick et al 2013;Fu et al 2013;Hsu et al 2013;Pattanayak et al 2013;Cho et al 2014). Recently, we and other groups independently presented several different methods for profiling genome-wide specificities of RGENs, which consist of gRNAs and the Cas9 protein originated from Streptococcus pyogenes, in human cells (Frock et al 2015;Kim et al 2015;Ran et al 2015;Tsai et al 2015;Wang et al 2015). All of these methods rely on high-throughput sequencing of human genomic DNA that is cleaved by RGENs in cells or in vitro.…”

“…All of these methods rely on high-throughput sequencing of human genomic DNA that is cleaved by RGENs in cells or in vitro. High-throughput, genomewide translocation sequencing (HTGTS) is based on translocations induced by nonhomologous end-joining (NHEJ) repair of two concurrent DNA double-strand breaks (DSBs) in cells (Frock et al 2015). 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.…”

Transcription-associated processes cause DNA double-strand breaks and translocations in neural stem/progenitor cells

“…Because of cellular heterogeneity in 3D genome organization (3,27,29), HTGTS detects, with great sensitivity, recurrent DSBs in any given genomic location (4,9,(26)(27)(28)(29) or recurrent classes of DSBs, such as DSBs at TSSs, that may occur at much lower frequency in any given location (ref. 9 and this study).…”

“…We previously developed an unbiased, high-throughput, genomewide, translocation sequencing (HTGTS) approach to identify DSBs via their translocation to bait DSBs at defined genomic sites in primary progenitor and activated mature B cells, NSPCs, and human cell lines (4,9,(26)(27)(28). Because of cellular heterogeneity in 3D genome organization (3,27,29), HTGTS detects, with great sensitivity, recurrent DSBs in any given genomic location (4,9,(26)(27)(28)(29) or recurrent classes of DSBs, such as DSBs at TSSs, that may occur at much lower frequency in any given location (ref.…”

“…HTGTS was performed and data were analyzed as described (4,9,27). In brief, libraries (500-1,000 bp fragment size) were purified and sequenced on the Illumina MiSeq platform.…”