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

• Published in: Genome Biology Vol. 24; no. 1; p. 85
• Main Authors: Marinov, Georgi K, Kim, Samuel H, Bagdatli, S Tansu, Higashino, Soon Il, Trevino, Alexandro E, Tycko, Josh, Wu, Tong, Bintu, Lacramioara, Bassik, Michael C, He, Chuan, Kundaje, Anshul, Greenleaf, William J
• Format: Journal Article
• Language: English
• Published: England BioMed Central 21-04-2023; BMC
• Subjects: CRISPR; CRISPR-Associated Protein 9 - genetics; CRISPR-Cas Systems; DNA, Single-Stranded - genetics; Editing; Enzymes; Epigenome; Gene Editing - methods; Gene mapping; Genome; Genomes; Method; Proteins; Single-stranded DNA
• ISSN: 1474-760X; 1474-7596; 1474-760X
• DOI: 10.1186/s13059-023-02930-z

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.