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For CRISPRa experiments, how specific are the gRNAs in targeting the gene of interest?
Several publications have shown CRISPRa to be highly specific by doing RNA seq expression analysis, but CRISPRa is a new technology and off-targeting still needs to be explored in more detail. Keep in mind that for CRISPRa off-targeting, the guide RNA needs to bind to the promoter region of another gene in order to have an off-target effect, which dramatically decreases the potential off-target space. Furthermore, the guide RNAs are designed based on a published algorithm that incorporates chromatin, nucleosome position, and sequence features to accurately predict highly effective guide RNAs and also applies a filter for off-target binding.
However, there might be examples of genes where the promoter region for one gene is in close proximity to another gene’s promoter region. Investigation of the genomic location for your gene of interest and performing expression analysis to confirm activation of the target gene without having effects on other proximal genes might be important for proper interpretation of the phenotypic analysis.