Figure 2.

Off-target detection methods simplified overview. (A) in silico algorithm prediction. Computer algorithms are utilized to predict off-target cleavage sites based on sequence similarities. The predicted sites are checked for off-target cleavage that may have occurred but does not allow detection of novel or random off-target sites. (B) in vitro selection. Partially randomized concatemeric DNA libraries are created via rolling-circle amplification of potential cleavage sites, which are checked for off-target cleavage. While a large library is created, the sequences are selected for by sequence similarities and may miss some off-target sites (C) Genome-wide assay. High-throughput sequencing of the entire genome is performed, essentially being an unbiased method as the entire genome is checked for off-target cleavage, although mosaicism may be missed.