The objective of this proposal is to develop a new chemical biology tool that integrates the CRISPR (clustered regularly interspaced short palindromic repeat) system with the CIP (Chemically Induced Proximity) method to achieve chemical or light inducible epigenome editing. Epigenetic modifications and chromatin structures are proposed to regulate gene activity. Epigenetic dysregulation has been linked to different human diseases including cancer. Although various observations associate specific epigenetic marks with certain gene activities, it is still difficult to establish the causal relationship, unless a specific and defined epigenetic environment can be artificially created in living cells in a locus-specific and spatiotemporal manner. New methods for such studies are needed as current methods for epigenetic research have their limitations to meet this goal. To address this, we propose to develop a novel inducible platform for epigenome editing that combines the CRISPR/dCas9 system with the CIP method. The CRISPR/dCas9 system has been shown to be a powerful genome targeting method when paired with unique sgRNAs (single guide RNAs) designed to target specific DNA sequences in the genome. We expect that this CRISPR/dCas9 system can be readily adapted to target chosen chromatin-modifying proteins to edit epigenetic landscape at chosen genomic loci. A chemical inducible system based on CRISPR/dCas9 system can be developed when combined with the CIP method. In the CIP system, a small molecule inducer triggers the association between two unique inducer-binding adaptor proteins that can be fused individually to two proteins of interest (e.g. dCas9 and a chromatin-modifying protein). We have developed a photo-activated CIP system that provides unique spatiotemporal controls. We envision that this new integrated CRISPR/CIP technology will allow using either small molecules or light to control the recruitment of chromatin-modifying proteins to specific genomic loci and enable the editing of local epigenetic environments with desired combination of epigenetic marks. We propose the following specific aims to test the new system and apply it to study the functions of specific epigenetic modifications.
Aim 1. Establish the chemical-inducible CRISPR-guided platforms to edit the epigenome.
Aim 2. Test light-inducible epigenome editing and investigate the interplay between specific histone modifications. After finishing this work, we expect to establish this unique tool that will significantly contribute to epigenetic research and potentially lead to the development of new therapies. We will also provide new information and insight regarding the roles and interactions between epigenetic marks.
Epigenetic modifications are proposed to control gene activities. Epigenetic dysregulation has been linked to various human diseases including cancer. Current methods are inadequate to dissect the functions of specific epigenetic marks and their interplays. To address this, we propose a new chemical biology method combining CRISPR and CIP technologies that allow the use of small molecules or light to achieve spatiotemporal-specific epigenome editing. We expect this new method will contribute to the research in the fields of epigenetics.
| Gao, Dan; Liang, Fu-Sen (2018) Chemical Inducible dCas9-Guided Editing of H3K27 Acetylation in Mammalian Cells. Methods Mol Biol 1767:429-445 |
| Zhao, Weiye; Nguyen, Huong; Zeng, Guihua et al. (2018) A chemically induced proximity system engineered from the plant auxin signaling pathway. Chem Sci 9:5822-5827 |
| Zeng, Guihua; Wang, Yi; Bruchez, Marcel P et al. (2018) Self-Reporting Chemically Induced Protein Proximity System Based on a Malachite Green Derivative and the L5** Fluorogen Activating Protein. Bioconjug Chem 29:3010-3015 |
| Chen, Tingjun; Gao, Dan; Zhang, Roushu et al. (2017) Chemically Controlled Epigenome Editing through an Inducible dCas9 System. J Am Chem Soc 139:11337-11340 |
