Delivery of Synthesized Entities to Specific Genomic Loci in Mammalian Cells via a Modified CRISPR/Cas9 System

Abstract

Histone post-translational modifications (PTMs) are chemical modifications that function as a combinatorial code to carefully orchestrate gene expression in eukaryotic organisms. This code leads to cellular function through mostly direct interactions with PTM reading, writing, and erasing enzymes beginning at the level of a single nucleosome. Aberrations in the code or the regulatory mechanisms are implicated in numerous human diseases, including oncogenesis. However, the causal role of these histone PTMs on in vivo recruitment of PTM effectors and crosstalk with other PTMs are difficult to tease apart with traditional biological tools that use only genetically accessible constructs. Advancements in synthetic peptide chemistry has allowed for atomic scale control of in vitro studies by directly characterizing the interactions between synthesized histone PTMs and relevant effectors in a simplified setting. In order to bridge the precise combinatorial control of synthetic chemistry to biologically appropriate sites and resolution, we have developed an approach that utilizes protein trans-splicing (PTS) to covalently join synthetic cargoes with Cas9, an expressed programmable gene-targeting platform. As a proof of concept, an exogenously prepared transcription-activating domain(VP64) was delivered and ligated in vivo on to Cas9 to directly increase transcription activation. Furthermore, this method was applied to study the direct effects of histone H3 polyacetylation on transcription on a specific gene locus. H3 polyacetylation has been correlated with actively transcribed genes but no causal relationship has been established. This method presents a general paradigm for localization of synthetic entities to specific genomic loci in mammalian cells for inquires in epigenetics and otherwise.