Strategies to Determine Off-Target Effects of Engineered Nucleases

Genome editing is greatly facilitated by using engineered nucleases to specifically cleave a pre-selected DNA sequence. Cellular repair of the nuclease-induced DNA breaks by either non-homologous end joining (NHEJ) or homology-directed repair (HDR) allows genome editing in a wide range of organisms and cell lines. However, if a nuclease cleaves at genomic locations other than the intended target, known as "off-target sites", it can lead to mutations, chromosomal loss or rearrangements, causing gain/loss of function and cytotoxicity. Although zinc finger nucleases (ZFNs), TAL effector nuclease (TALENs), and CRISPR/Cas9 systems have been used successfully to create specific DNA breaks in cells, they lack perfect specificity and may result in off-target cleavage. Methods have been developed to predict and to quantify the off-target cleavage events, which are very important for optimizing nuclease design and determining if the gene editing approaches are highly specific. These methods have the potential to significantly facilitate the design of engineered nucleases for genome editing applications.