CRISPR/Cas9 has revolutionized the ability to edit cellular DNA and is poised to transform the treatment of genetic diseases. One of the major concerns regarding its therapeutic use is the potential for off-target DNA cleavage, which could have detrimental consequences in vivo. To circumvent this, a number of strategies have been employed to develop next-generation CRISPR/Cas9 systems with improved specificity. These include the development of new protein variants of Cas9, as well as chemically modified guide RNA molecules. Here, we provide detailed protocols for two in vitro methods that enable the specificity of first- and next-generation CRISPR/Cas9 systems to be compared, and we demonstrate their applicability to evaluating chemically modified guide RNAs. One of these assays allows the specificity of different guide RNA/Cas9 complexes to be compared on a set of known off-target DNA sequences, while the second provides a broad specificity profile based on cleavage of a massive library of potential off-target DNA sequences. Collectively, these assays may be used to evaluate the specificity of different CRISPR/Cas9 systems on any DNA target sequence in a time- and cost-effective manner.
Keywords: CRISPR/Cas9; Cas9 variants; Chemically modified guide RNAs; DNA cleavage specificity; Gene editing; Genome engineering; In vitro DNA cleavage assay; In vitro high-throughput specificity profiling assay; Next-generation CRISPR/Cas9.