Development of CRISPR-Cas tools towards rewiring 3D genome structure

Abstract

New genomic methods have revealed a high level of spatial organization in the nucleus, and the subnuclear positioning of genes correlates with transcriptional activity. It is thought that 3D genome organization and positioning directly influences transcriptional activity – perhaps by re-positioning genes near distal enhancers or to areas high in concentration of epigenetic modifiers. Limited studies in both human and yeast cells have revealed that gene repositioning affects transcription in some, but not all, genomic contexts. These studies raise the question: What rules govern transcriptional sensitivity to spatial re-positioning mechanisms? Such a question has remained elusive as conventional methodologies to rewire gene positioning rely on integration of a DNA binding site at every locus of interest. This approach stymies genomic screens involving multiple loci, particularly in human cells, where genome engineering remains difficult. Use of the CRISPR-Cas system enables researchers to target endogenous sites, bypassing the need for genome modification, which opens the door to reprogram the location of endogenous genes. In this work, I demonstrate two advances to the genome re-organization field. First, we show that a programmable CRISPR-Cas system can be used to localize genes to the nuclear periphery in budding yeast. Second, we demonstrate the development of a CRISPR-Cas allosteric sensor of DNA binding which might be fundamental to forming efficient long- range loops. These technological advancements may aid researchers to assess the functional consequences of gene repositioning.