Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases
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CitationFrock, Richard L., Jiazhi Hu, Robin M. Meyers, Yu-Jui Ho, Erina Kii, and Frederick W. Alt. 2014. “Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases.” Nature biotechnology 33 (2): 179-186. doi:10.1038/nbt.3101. http://dx.doi.org/10.1038/nbt.3101.
AbstractAlthough 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.
Citable link to this pagehttp://nrs.harvard.edu/urn-3:HUL.InstRepos:21462344
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