Direct profiling of genome-wide dCas9 and Cas9 specificity using ssDNA mapping (CasKAS)
Marinov, G. K.; Kim, S. H.; Bagdatli, S. T.; Trevino, A. E.; Tycko, J.; Wu, T.; Bintu, L.; Bassik, M. C.; He, C.; Kundaje, A.; Greenleaf, W. J.
Show abstract
Detecting and mitigating off-target activity is critical to the practical application of CRISPR-mediated genome and epigenome editing. While numerous methods have been developed to map Cas9 binding specificity genome-wide, they are generally time-consuming and/or expensive, and not applicable to catalytically dead CRISPR enzymes. We have developed a rapid, inexpensive, and facile assay for identifying off-target CRISPR enzyme binding and cleavage by chemically mapping the unwound single-stranded DNA structures formed upon binding of a sgRNA-loaded Cas9 protein ("CasKAS"). We demonstrate this method in both in vitro and in vivo contexts.
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