Genomic targeting of epigenetic probes using a chemically tailored Cas9 system

Significance Programmable DNA-binding proteins such as nuclease-deficient Cas9 (dCas9) offer a simple system for genomic localization of genetically encodable biomolecules. These tools have enabled characterization of numerous different chromatin effectors at specific genetic elements. The delivery of fully synthetic cargos to specific loci is currently limited by a lack of adequate technologies. Here we used protein trans-splicing to ligate chemical moieties to dCas9 in vitro and subsequently deliver these molecules to live cells. We demonstrate the effectiveness and versatility of this method by delivering both small-molecule and proteinaceous epigenetic probes to endogenous genomic sites where they interact with their target proteins. This facile and modular strategy will find broad use in locus-specific targeting of chemical cargos to study nuclear processes. Recent advances in the field of programmable DNA-binding proteins have led to the development of facile methods for genomic localization of genetically encodable entities. Despite the extensive utility of these tools, locus-specific delivery of synthetic molecules remains limited by a lack of adequate technologies. Here we combine the flexibility of chemical synthesis with the specificity of a programmable DNA-binding protein by using protein trans-splicing to ligate synthetic elements to a nuclease-deficient Cas9 (dCas9) in vitro and subsequently deliver the dCas9 cargo to live cells. The versatility of this technology is demonstrated by delivering dCas9 fusions that include either the small-molecule bromodomain and extra-terminal family bromodomain inhibitor JQ1 or a peptide-based PRC1 chromodomain ligand, which are capable of recruiting endogenous copies of their cognate binding partners to targeted genomic binding sites. We expect that this technology will allow for the genomic localization of a wide array of small molecules and modified proteinaceous materials.