Cell-Type-Specific CRISPR Activation with MicroRNA-Responsive AcrllA4 Switch

Anti-CRISPR proteins have the potential to regulate CRISPR-Cas systems in a cell-type-specific manner. To selectively edit the genome in target cells, we controlled the expression of AcrllA4, a Streptococcus pyogenes Cas9 inhibitor, based on endogenous microRNA (miRNA) activity. We designed a miRNA-responsive AcrllA4 switch, which is a synthetic mRNA that contains a completely complementary sequence to an arbitrary miRNA at the 5′-UTR region and encodes AcrllA4. Together with the Cas9- or dCas9-VPR-guide RNA complex, this switch functions as a cell-specific Cas9 or dCas9-VPR activator that induces gene knockout or activation depending on the target miRNA. By sensing intracellular miRNAs, the conditional CRISPR-Cas9 ON system that we report could provide a powerful tool for future therapeutic applications and genome engineering.