# Optimization of a massively parallel genome editing approach to link regulatory elements to their target genes during mouse germ layer formation

> **NIH NIH F31** · UNIVERSITY OF WASHINGTON · 2024 · $56,974

## Abstract

Cell fate diversification is part and parcel to mammalian development. With the completion of the Human
Genome Project and subsequent large-scale efforts to functionally characterize the genome, we now know
that coding and non-coding DNA interdependently affect cell fate decisions. High-throughout, largely
descriptive assays carried out by the Encyclopedia of DNA Elements (ENCODE) Project and others like it,
have contributed to an increasingly curated list of DNA and RNA regulatory elements associated with cell-type
specific transcriptional and epigenetic programs. However, while it is estimated that 1 million cis-regulatory
elements, presumably mostly enhancers, regulate the protein-coding genome, their identity and
the genes they regulate remain largely unknown.
Here, I propose to apply highly scalable functional approaches to candidate enhancer elements in a
developmentally relevant and tractable system. Specifically, I am further developing a framework, known
as 'massively parallel genome editing' or MPGE, that utilizes a CRISPR/Cas9 screening approach and
single-cell RNA sequencing (scRNA-seq) to globally capture perturbations to gene expression. I will apply
this method to mouse embryonic stem cell-derived germ layers, which are essential for body plan assembly
in early development. Together, this study design will validate candidate enhancer elements in their native
context while also identifying the target gene(s) that they regulate. This functional validation of germ layer-specific
enhancer-gene pairs will yield insights into how cell fates emerge in early development.

## Key facts

- **NIH application ID:** 10794974
- **Project number:** 5F31HG011576-04
- **Recipient organization:** UNIVERSITY OF WASHINGTON
- **Principal Investigator:** Samuel Regalado
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $56,974
- **Award type:** 5
- **Project period:** 2021-03-16 → 2025-03-15

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10794974

## Citation

> US National Institutes of Health, RePORTER application 10794974, Optimization of a massively parallel genome editing approach to link regulatory elements to their target genes during mouse germ layer formation (5F31HG011576-04). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10794974. Licensed CC0.

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