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Nat Biotechnol. 2015 Feb;33(2):179-86. doi: 10.1038/nbt.3101. Epub 2014 Dec 15.

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

Author information

1
1] Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA. [2] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. [3] Howard Hughes Medical Institute, Boston, Massachusetts, USA.

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.

PMID:
25503383
PMCID:
PMC4320661
DOI:
10.1038/nbt.3101
[Indexed for MEDLINE]
Free PMC Article

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