Epigenetic modifications of histone and DNA control gene expression and critically shape phenotypes and cell states. These modifications are tightly controlled by interactions with a constellation of trans-acting regulatory factors, and are dysregulated in a plethora of diseases. Next-generation sequencing (NGS) technologies have allowed genome-wide profiling of these modifications in diverse cell types, normal and disease conditions as well as across individuals. However, these strategies have yielded mostly associative and/or correlative insight into relationships between epigenetic state, gene expression and phenotype with limited power to establish the causality of individual epigenetic modifications that is fundamental to understanding normal physiology and diseases. Existing techniques to investigate phenotypic consequences of epigenetic gene regulation irreversibly delete stretches of genomic sequence that can have multiple functions, or perturb protein factors that control thousands of genes, confounding accurate conclusion. A scalable toolbox allowing in situ and in vivo combinatorial modifications of epigenetic states would offer unprecedented opportunities for both mechanistic studies and unbiased discovery of novel functional elements at the genomic scale. This project will respond to this need by developing an expandable molecular toolkit to precisely and reversibly manipulate defined epigenetic modifications (e.g., H3K27ac) at defined genomic addresses based on our innovative CRISPR/Casilio platform. There are three Specific Aims in this project.
Aim 1 is focused on the development of a comprehensive set of epigenetic editing modules with which Casilio can achieve multiplexed and combinatorial edits, whereby different epigenetic modifications can be elicited simultaneously at distinct genomic loci while multiple modifications can also be induced in each locus.
Aim 2 will deliver Casilio-enabled cell lines with which scientists can achieve epigenome editing with unprecedented ease by delivering short RNA guides.
Aim 3 will center on the development of genome-wide guide libraries targeting genomic regulatory elements such as enhancers and insulators with which scientists can perform reverse epigenetic screens to discover novel elements and epigenetic modifications causative to phenotype. This toolkit, once developed, will transform the ways we study epigenetics by providing a fine and scalable technique to directly edit epigenetic states at defined targets, to investigate the underlying causes of gene regulatory changes observed in biological processes and diseases. To truly benefit the field of functional genomics, we will share our methodology and reagents at every stage of platform development with the scientific community. Through the establishment of standards and a module registry as well as reagent sharing via an open repository, we hope to create a sustainable ecosystem of Casilio module users and developers to apply and expand the Casilio toolbox for epigenetic editing. Due to the programmability and precision, epigenetic editing enabled by Casilio may provide new means and tools for powering personalized and precision medicine.

Public Health Relevance

Changes to the way genes are expressed, a phenomenon known as epigenetic regulation, can deeply affect cell function, health and disease. The goal of this project is to develop state-of-the-art tools to more precisely manipulate epigenetic regulation. Such tools will enable researchers to establish causal links between specific epigenetic changes and their biological effects, and ultimately could represent a new means of therapeutic intervention for precision medicine.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
1R01HG009900-01
Application #
9424125
Study Section
Genomics, Computational Biology and Technology Study Section (GCAT)
Program Officer
Pazin, Michael J
Project Start
2018-01-01
Project End
2022-12-31
Budget Start
2018-01-01
Budget End
2018-12-31
Support Year
1
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Jackson Laboratory
Department
Type
DUNS #
042140483
City
Bar Harbor
State
ME
Country
United States
Zip Code
04609