In particular, nuclease-deactivated Cas (dCas) proteins have been applied for use of controlling the gene expression via directed dCas binding and the localization of domains driving gene up- and downregulation (termed CRISPRa and CRISPRi, respectively). The adoption of CRISPR systems has enabled a wide range of synthetic biology applications by allowing the rapid targeting of CRISPR-associated (Cas) proteins to almost anywhere in the genome via a guide RNA (gRNA). This system should allow for the development of novel epigenome editing tools which will be useful in a wide array of biological research and engineering applications. We report the creation of single-protein CRISPR constructs bearing combinations of three epigenetic editing domains, termed KAL, that can stably repress the gene expression. We characterize the dynamics of durable gene silencing and reactivation, as well as the induced epigenetic changes of this system. Here, we describe the design of a reporter system for quantifying the ability of CRISPR epigenome editors to produce a stable gene repression. Development of CRISPR-based epigenome editing tools is important for the study and engineering of biological behavior.
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