Developing Single-Cell Functional Genomic Technologies to Investigate DNA Methylation Changes in Human Diseases

Aberrant DNA methylation is linked to a wide range of human diseases. Over the last decade, numerous sites have been identified where DNA methylation changes correlate with disease pathology. However, establishing the causality between them has been challenging. This is mainly due to the lack of functional assays that introduce site-specific DNA methylation changes and observe subsequent outcomes to infer their functions directly. To address this, I developed a high-throughput assay, methMPRA, to quantify the causal effects of DNA methylation on gene regulatory activities. Integrating methMPRA with single-cell sequencing and CRISPR-based methylation editing would allow a comprehensive dissection of the causal relationship between DNA methylation and gene expression. Thus, I propose to develop two single-cell functional assays (Aim 1, K99): (1) develop single-cell methMPRA to test how DNA methylation affects gene regulatory activities. I will modify methMPRA into a single-cell format using a dual-barcode system and generate a catalog of methylation-sensitive regulatory elements in a cell type-specific manner. (2) develop single-cell CRISPR methylation screen to assess the effect on gene expression. I will introduce disease-related methylation changes in their native genomic locations and observe subsequent gene expression changes through single-cell RNA- seq. This will pinpoint the genes affected by DNA methylation perturbations and quantify the effect on gene expression. As a proof-of-concept, I will study cancer-associated methylation changes and perform the single-cell assays in a mixture of tumor cell lines. In Aim 2 (R00), I will develop a novel method, Perturb-MethylSeq, to understand how abnormal DNA methylation patterns are established during pathogenesis. This method involves multiplexed gene knockdown and measuring subsequent changes in DNA methylation profiles using single-cell methylation sequencing. I will apply this method to identify genes essential for establishing abnormal DNA methylation patterns during tumorigenesis. Together, these aims will introduce a versatile toolkit to study the functional roles and mechanistic underpinnings of DNA methylation changes in various biological processes. To accomplish these aims, I will have additional training supported by my primary mentor, Dr. Nadav Ahituv (functional genomics), co-mentor Dr. Jay Shendure (genetics and genomics), and my collaborators, Dr. Martin Kircher (bioinformatics) and Dr. Chongyuan Luo (single-cell epigenomics). My career development plan includes training in single-cell sequencing and CRISPR screening. UCSF is known for its interactive environment, providing all the facilities needed for the proposed research and fostering an intellectual atmosphere for collaborations. Together, the training in the K99 phase will facilitate my transition to an independent career focusing on functional genomic technology and epigenetic etiology. Project Number: 1K99HG014204-01 | Fiscal Year: 2025 | NIH Institute/Center: National Human Genome Research Institute (NHGRI) | Principal Investigator: Chengyu Deng | Institution: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO, SAN FRANCISCO, CA | Award Amount: $135,206 | Activity Code: K99 | Study Section: Genome Research Study Section[GNOM-G] View on NIH RePORTER: https://reporter.nih.gov/project-details/11110680