An integrative genomics toolkit for rewriting regulatory DNA to reprogram gene expression

About This Grant

PROJECT SUMMARY Regulatory DNA elements, including enhancers and promoters, encode multiple transcription factor binding sites (TFBS) that quantitatively tune gene expression in a cell-type specific fashion. Understanding and engineering regulatory DNA could unlock new therapeutic approaches — for example, to restore proper expression of a disease gene. Yet, we have lacked computational models to predict which sequence changes to regulatory DNA will have which effects on gene expression as well as lacked experimental tools to test these predictions in a sufficiently scalable fashion. Our team has now developed innovative tools to dramatically increase the throughput of characterizing edits to regulatory DNA — including computational tools to optimize sequences to achieve desired gene expression outcomes, and experimental tools to test their impact on gene expression in high-throughput screens. We will further develop and apply this toolkit in the context of studying 4 genes for different diseases where genome editing therapies could be transformative. In Aim 1, we will use CRISPR prime editing to rewrite TFBS in gene promoters to achieve quantitative tuning of gene expression. In Aim 2, we will modulate gene expression through insertion of libraries of candidate enhancers and silencers at defined distances from gene promoters. In Aim 3, we will engineer sequence edits in cardiac organoids and measure the cell type specificity of their effects using novel single-cell readout approaches. Together, these studies will (i) illuminate basic mechanisms of gene regulation, (ii) map the programmability of 5 disease genes with distinct properties, and (iii) hone a novel integrative genomics toolkit to design and test edits to regulatory DNA to achieve specific gene expression outcomes. This approach will advance our understanding of genome regulation and lay the foundations for rational approaches to reprogram gene expression to treat many types of human diseases.