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

Abstract

Although great progress has been made in the characterization of the 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 hotspot numbers 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 more than tenfold. We also identified translocations between bona fide nuclease targets on homologous chromosomes, an undesired collateral effect that has not been described previously. Finally, HTGTS confirmed that the Cas9D10A paired nickase approach suppresses off-target cleavage genome-wide.

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Figure 1: LAM-PCR HTGTS from Cas9:RAG1A on-target and off-target bait sites.
Figure 2: Distinct prey break-site junction joining to homologous chromosomes.
Figure 3: Cas9n paired nick DSB HTGTS libraries.
Figure 4: Identification of off-target sites from other nucleases using the universal Cas9:RAG1B bait.
Figure 5: Nuclease titration to measure on-target/background efficiency.

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Acknowledgements

This work is supported by the National Institutes of Health Grants P01CA109901 and P01AI076210. R.L.F. was supported by the National Institutes of Health NRSA T32CA070083 and is supported by the National Health Institutes of Health NRSA T32AI007512. J.H. is supported by Robertson Foundation/Cancer Research Institute Irvington Fellowship. F.W.A. is an investigator of the Howard Hughes Medical Institute. A549 cells were provided by David Weinstock (Dana-Farber Cancer Institute).

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R.L.F., J.H. and F.W.A. designed the research; R.L.F., J.H. and E.K. performed the research; R.L.F., J.H., R.M.M., Y.-J.H. and E.K. analyzed the data; R.L.F. and F.W.A. wrote the paper.

Corresponding author

Correspondence to Frederick W Alt.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–16 and Supplementary Tables 1–8 (PDF 9006 kb)

Supplementary Dataset 1

Hotspot listing for HTGTS baits (XLSX 285 kb)

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Frock, R., Hu, J., Meyers, R. et al. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nat Biotechnol 33, 179–186 (2015). https://doi.org/10.1038/nbt.3101

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