# Dissecting interactions across gene regulatory layers in single cells

> **NIH NIH R35** · SOUTHERN METHODIST UNIVERSITY · 2022 · $360,076

## Abstract

ABSTRACT
Biological processes are controlled by multiple genes working in concert to achieve a given function. This
phenomenon is apparent in genetic interactions, defined as a phenotype observed in a double mutant not
easily explained by the phenotypes in the respective single mutants. While genetic interactions have long
been recognized as important drivers of animal phenotypes, it has not been possible to perform genetic
interaction analysis in animals in a systematic, null allele, reverse-genetics fashion. This is a critical gap,
because understanding healthy and disease states in animals requires an appreciation of how multiple genes
coordinately affect a given phenotype. To overcome this gap, we have developed a CRISPR/Cas9 toolkit that
enables targeted genome modification and subsequent genetic interaction analysis in the nematode worm
Caenorhabditis elegans, thus enabling for the first time systematic targeted genetic interaction profiling in
animals. We will focus on genetic interactions among factors regulating gene expression. Proper gene
expression is controlled by multiple layers of regulation (e.g. transcription, RNA processing, translation) but
little is known about how these layers are coordinated at the level of single cells. The first direction of the lab
therefore is to profile genetic interactions between different layers of gene expression, specifically focusing on
transcription factors (TFs) and RNA binding proteins (RBPs). Double mutant combinations with unexpected
phenotypes will be the entry point to mechanistic understanding of how combinations of TFs and RBPs
coordinately control gene expression. The second direction of the lab will be to understand the regulation of
alternative splicing at the single cell level by combinations of TFs and RBPs. Individual cell types can be
defined by the presence of TFs and the resulting gene expression patterns, but can also be further refined by
the presence of splicing factors and the resulting isoforms expressed. We have created a large number of in
vivo splicing reporters in C. elegans and found extensive alternative splicing at the single cell level. Using a
combination of forward and reverse genetics we have identified a number of splicing factors, as well as a
surprising number of TFs, important for specific alternative splicing regimes at the single cell level. We now
plan to investigate the mechanisms by which these factors combine to control splicing at the single cell level,
as well as the functional consequences of such splicing. Together these directions will represent a key
advance in our understanding of combinatorial action of gene regulatory factors and how they coordinately
ensure proper gene expression.

## Key facts

- **NIH application ID:** 10428588
- **Project number:** 5R35GM133461-04
- **Recipient organization:** SOUTHERN METHODIST UNIVERSITY
- **Principal Investigator:** Adam Norris
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $360,076
- **Award type:** 5
- **Project period:** 2019-08-01 → 2024-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10428588

## Citation

> US National Institutes of Health, RePORTER application 10428588, Dissecting interactions across gene regulatory layers in single cells (5R35GM133461-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10428588. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*