Direct profiling of genome-wide dCas9 and Cas9 specificity using ssDNA mapping (CasKAS)

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 genome-wide sgRNA 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 binding and cleavage by chemically mapping the unwound single-stranded DNA structures formed upon CRISPR binding (``CasKAS''). We demonstrate this method in both in vitro and in vivo contexts. We have developed a rapid, inexpensive, and facile assay for identifying off-target CRISPR binding and cleavage by chemically mapping the unwound single-stranded DNA structures formed upon CRISPR binding (``CasKAS'').