CasKAS: direct profiling of genome-wide dCas9 and Cas9 specificity using ssDNA mapping

作者：Georgi K. Marinov, Samuel H. Kim, S. Tansu Bagdatli, Soon Il Higashino, Alexandro E. Trevino, Josh Tycko, Tong Wu, Lacramioara Bintu, Michael Cory Bassik, Chuan He, Anshul Bharat Kundaje, William James Greenleaf · 发表于：Genome biology · 年份：2023 · DOI：10.1186/s13059-023-02930-z · 被引用次数：13 · 研究领域：CRISPR and Genetic Engineering、Advanced biosensing and bioanalysis techniques、RNA and protein synthesis mechanisms

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.