Figure 8.

Off-target discovery and validation process.

The first step in the off-target identification process is discovery, either through in silico prediction of similar sequences, in vitro activity of the nuclease on naked or genomic-context DNA, or a combination of both in silico and in vitro methods. These methods produce a list of putative off-target sites that can then be further interrogated by targeted PCR amplification of the candidate sites in the cell type of interest, followed by next generation sequencing. Sites containing indels are considered verified off-targets.