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.

In vivo epigenome editing and transcriptional modulation using CRISPR technology

“…In contrast, HDAC3 functions as a corepressor by deacetylating the H3K27ac on enhancer elements (Hatzi et al 2013). Recently, CRISPR technology has also been successfully used to recruit endogenous chromatin regulators to targeted genomic loci (Braun et al 2017; Liszczak et al 2017) or manipulation of nuclear architecture and chromatin looping (Hao et al 2017; Morgan et al 2017). However, most of these studies have not yet been tested in vivo.…”

“…To enable more robust epigenome editing and transcriptional modulation using AAV-CRISPR system, dCas9 conjugation through protein trans-splicing and dimerizing techniques can be adapted to recruit large endogenous chromatin regulators to specific genomic loci (Braun et al 2017; Liszczak et al 2017) (Figure 5). For example, Fkbp/Frb inducible recruitment for epigenome editing by dCas9 (FIRE-dCas9) system was a recently developed dCas9 conjugation approach that has been used to achieve rapid and reversible recruitment of endogenous chromatin regulators to specific genomic loci (Braun et al 2017).…”

“…By recruiting mSWI/SNF (BAF) complex to bivalent promoters, transcription activation was induced through the loss of repressive H3K27me3 and increase in active H3K4me3 marks. A chemically tailored dCas9 system was also recently developed by using protein trans-splicing to ligate synthetic elements (IntC-cargo such as IntC-JQ1 and IntC-UNC3866) to a dCas9-IntN (Liszczak et al 2017). The resulting dCas9-IntN:IntC-cargo fusion was capable of recruiting endogenous copies of their cognate binding partners (e.g.…”

Emerging Chemistry Strategies for Engineering Native Chromatin

“…29,30 Thus, as we write this piece, many dozens of modified histones have been generated using chemistry-driven methods. These reagents have been used to study various aspects of chromatin biochemistry, 31,32 in many cases allowing mechanistic hypotheses generated from cell-based genomic data to be tested.…”

Efficient and Innocuous Live‐Cell Delivery: Making Membrane Barriers Disappear to Enable Cellular Biochemistry

“…Alternatively, fully synthetic histones modified by PTMs using several of the chemistry‐based labeling strategies currently available could be delivered into cells directly, assuming proper integration into nucleosomes postdelivery . Alternatively, the nuclease‐deficient Cas9 (dCas9) can be modified synthetically to display chemical or proteinaceous epigenetic probes . Upon complex formation between and a guide RNA and upon subsequent successful delivery into live cells, such constructs are directed to specific genomic localizations.…”