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CasKAS: direct profiling of genome-wide dCas9 and Cas9 specificity using ssDNA mapping
Georgi K Marinov1, Samuel H Kim2, S Tansu Bagdatli3
1Department of Genetics, School of Medicine, Stanford University, Stanford, CA, 94305, USA. GKM359@gmail.com.
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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 CasKAS, 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. We demonstrate this method in both in vitro and in vivo contexts.
