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

ABSTRACT: 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.

SUBMITTER: Marinov GK

PROVIDER: S-EPMC10120127 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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

Marinov Georgi K GK Kim Samuel H SH Bagdatli S Tansu ST Higashino Soon Il SI Trevino Alexandro E AE Tycko Josh J Wu Tong T Bintu Lacramioara L Bassik Michael C MC He Chuan C Kundaje Anshul A Greenleaf William J WJ

Genome biology 20230421 1

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- ...[more]

PMID: 37085898

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

Project description: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.

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