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. 2013 Oct;41(19):e181.
doi: 10.1093/nar/gkt716. Epub 2013 Aug 14.

In silico abstraction of zinc finger nuclease cleavage profiles reveals an expanded landscape of off-target sites

Jeffry D Sander  1 , Cherie L Ramirez, Samantha J Linder, Vikram Pattanayak, Noam Shoresh, Manching Ku, Jennifer A Foden, Deepak Reyon, Bradley E Bernstein, David R Liu, J Keith Joung
Affiliations

In silico abstraction of zinc finger nuclease cleavage profiles reveals an expanded landscape of off-target sites

Jeffry D Sander et al. Nucleic Acids Res. 2013 Oct.

Abstract

Gene-editing nucleases enable targeted modification of DNA sequences in living cells, thereby facilitating efficient knockout and precise editing of endogenous loci. Engineered nucleases also have the potential to introduce mutations at off-target sites of action. Such unintended alterations can confound interpretation of experiments and can have implications for development of therapeutic applications. Recently, two improved methods for identifying the off-target effects of zinc finger nucleases (ZFNs) were described-one using an in vitro cleavage site selection method and the other exploiting the insertion of integration-defective lentiviruses into nuclease-induced double-stranded DNA breaks. However, application of these two methods to a ZFN pair targeted to the human CCR5 gene led to identification of largely non-overlapping off-target sites, raising the possibility that additional off-target sites might exist. Here, we show that in silico abstraction of ZFN cleavage profiles obtained from in vitro cleavage site selections can greatly enhance the ability to identify potential off-target sites in human cells. Our improved method should enable more comprehensive profiling of ZFN specificities.

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Figures

Figure 1.
Figure 1.
Schematic illustrating the original method by Pattanayak et al. (8) (blue arrows) and the enhanced approach that incorporates addition of a classifier-based step (green arrows).
See this image and copyright information in PMC

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