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

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 cat...

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Bibliographic Details
Published in:Genome Biology 2023-04, Vol.24 (1), p.85-85, Article 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: Article
Language:English
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
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Summary: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.
ISSN:1474-760X
1474-7596
1474-760X
DOI:10.1186/s13059-023-02930-z