Abstract
The CRISPR/Cas9 system has transformed how gene knockout and knock-in studies are performed in the lab, and it is poised to revolutionize medicine. However, one of the present limitations of this technology is its imperfect specificity. While CRISPR/Cas9 can be programmed to cut a specific DNA target sequence with relative precision, off-target sequence cleavage can occur in large genomes. Importantly, several techniques have recently been developed to measure CRISPR/Cas9 on- and off-target DNA cleavage in cells. Here, we present detailed protocols for evaluating the specificity of CRISPR/Cas9 and related systems in cells using both targeted-approaches, in which off-target sites are known a priori, and unbiased approaches which are able to identify off-target cleavage events throughout an entire genome. Together, these techniques can be used to assess the reliability of experimental models generated using CRISPR/Cas9 as well as the safety of therapeutics employing this technology.
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