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. Author manuscript; available in PMC: 2015 May 21.
Published in final edited form as: Methods Enzymol. 2014;546:47–78. doi: 10.1016/B978-0-12-801185-0.00003-9

Figure 1. Overview of methods to study the specificity of nucleases.

Figure 1

Potential substrate sequences of interest (colored strands) are subjected to nuclease cleavage to identify cleaved sequences (broken red and orange strands). In discrete off-target site assays, sequences are individually subjected to nuclease cleavage in a low- or high-throughput manner. In genome-wide selections, a few potential off-target sites are cleaved within predominantly uncleaved genomic DNA (black strands) and detected by viral integration. Using in vitro selection, many potential off-target sites in a vast DNA library are selected for binding or for their ability to be bound or cleaved site-specific nucleases in vitro.