CasKAS: direct profiling of genome-wide dCas9 and Cas9 specificity using ssDNA mapping.
Georgi K MarinovSamuel H KimS Tansu BagdatliSoon Il HigashinoAlexandro E TrevinoJosh TyckoTong WuLacramioara BintuMichael C BassikChuan HeAnshul KundajeWilliam J GreenleafPublished in: Genome biology (2023)
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.
Keyphrases
- crispr cas
- genome wide
- genome editing
- dna methylation
- binding protein
- high resolution
- dna binding
- copy number
- high density
- drug delivery
- gene expression
- circulating tumor
- single molecule
- single cell
- loop mediated isothermal amplification
- structural basis
- transcription factor
- cancer therapy
- quantum dots
- nucleic acid
- gold nanoparticles
- small molecule
- reduced graphene oxide
- metal organic framework
- circulating tumor cells