Diagenode

Optimize the selection of guide RNA by ChIP to keep CRISPR on-target

The mechanisms of target recognition and target specificity of the Cas9 protein is still not completely understood. A major hurdle of this technology is the introduction of double-strand breaks (DSBs) at sites other than the intended on-target site (off-target effects). All CRISPR/Cas9 applications require the verification of the specific binding of the sgRNA at the locus of interest. Chromatin immunoprecipitation followed by real-time PCR (ChIP-qPCR) is a technique of choice for studying protein-DNA interactions. In this study, we show a successful ChIP-qPCR method to verify the binding efficiency of the dCas9/sgRNA complex in the targeted region; and ChIP-seq – to monitor off-target bindings of the dCas9/sgRNA complex in the genome.

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