title: Genomic targeting of epigenetic probes using a chemically tailored Cas9 system.
abstract: 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.
authors.affiliation: Department of Chemistry, Princeton University, Princeton, NJ 08544.
authors.forename: Glen P
authors.initials: GP
authors.lastname: Liszczak
authors.affiliation: Department of Chemistry, Princeton University, Princeton, NJ 08544.
authors.forename: Zachary Z
authors.initials: ZZ
authors.lastname: Brown
authors.affiliation: Department of Chemistry, Princeton University, Princeton, NJ 08544.
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Kim
authors.affiliation: Department of Chemistry, Princeton University, Princeton, NJ 08544.
authors.forename: Rob C
authors.initials: RC
authors.lastname: Oslund
authors.affiliation: Department of Chemistry, Princeton University, Princeton, NJ 08544.
authors.forename: Yael
authors.initials: Y
authors.lastname: David
authors.affiliation: Department of Chemistry, Princeton University, Princeton, NJ 08544 muir@princeton.edu.
authors.forename: Tom W
authors.initials: TW
authors.lastname: Muir
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D064130:CRISPR-Associated Proteins / Q000378:metabolism*; D064113:CRISPR-Cas Systems / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D057890:Epigenomics / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D023281:Genomics / Q000379:methods; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D064130:CRISPR-Associated Proteins; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins
keywords: chemical biology; epigenome engineering; intein splicing; protein delivery; protein semisynthesis