Enhanced Epigenome Editing Technologies for Controlling Mammalian Gene Expression

About This Grant

PROJECT SUMMARY Genome editing is rapidly transforming biology and medicine by enabling the precise modification of DNA sequences in vivo. The technologies used for genome editing have also provided a means to modulate the expression of a target gene by facilitating the recruitment of transcriptional effector domains to a target site. One such tool capable of recreating the native mechanisms of gene activation consists of catalytically inactive Cas9 (dCas9) fused with the histone acetyltransferase p300, which, by acetylating histones near a target site, can activate gene expression through a nearly physiological approach. Nonetheless, current dCas9-p300 systems are inefficient, unpredictable, and incompatible with adeno-associated virus (AAV) delivery, which hinders future applications of this technology. Here, we propose to integrate state-of-the-art techniques including protein engineering, machine learning, and viral-vector design to create an innovative toolkit of epigenome editors that can be used to activate gene expression in vivo. Aim 1 will leverage phylogenetic diversity and directed evolution in mammalian cells to identify p300 variants with enhance gene activation capabilities. In Aim 2, we will develop machine-learning models that integrate target-site sequence features and nucleosome architecture to predict actionable target sites for efficient acetylation and gene activation. And, in Aim 3, we will engineer split-intein and ultracompact epigenome editors that can be packaged within single or dual AAV vectors to enable in vivo gene activation. To accomplish these objectives, we have assembled a multidisciplinary team with collective expertise in epigenome editing (Dr. Perez-Pinera), computational biology (Dr. Song) and AAV gene delivery (Dr. Gaj). Our collaborative efforts will: (1) yield a toolkit of programmable, tunable, and deliverable gene-activation tools, (2) facilitate the discovery of fundamental principles for epigenome editor design, and (3) enable the development of therapeutic applications for multiple disorders including developmental and metabolic disorders. We anticipate that the innovative and interdisciplinary nature of this proposal will yield technologies that will broadly impact biotechnology and medicine.