# Manipulating and Interrogating Spatial Transcriptomics

> **NIH NIH DP1** · STANFORD UNIVERSITY · 2023 · $1,080,800

## Abstract

ABSTRACT
Spatial mRNA organization plays a fundamental role in diverse cellular processes and disease. In large,
compartmentalized cells (e.g., neurons and embryos), subcellular mRNA localization offers a core mechanism
for the spatiotemporal regulation of protein synthesis. Since the initial discovery of subcellular mRNA distribution
in 1983, high-throughput imaging and sequencing methods have revealed that, in many cell types, thousands of
RNAs are localized to distinct compartments. For example, many axonal-related mRNAs in neurons will transport
to the “site of needed” along the very long (>100μm) axon, which likely play an important role in axon
development and local synaptic activities. Furthermore, mounting evidence shows a correlation between
aberrant spatial RNA organization and an increasing number of diseases, including amyotrophic lateral sclerosis
(ALS), fragile X syndrome (FXS), and spinal muscular atrophy (SMA). However, due to a lack of technologies
that allow for the tracking and manipulation of the spatial localization of endogenous mRNAs in primary cells and
in vivo, the mechanism and functional relevance of spatial organization has only been explored for a small
number of mRNAs. In this proposal, we seek to establish a set of technologies as a new foundation to study
spatial RNA biology, by developing an integrated framework that allows for sophisticated computational analysis,
real-time RNA tracking, and programmable spatial manipulation of any endogenous mRNA(s) in situ and in vivo,
on a high-throughput (>1,000 mRNAs in parallel) scale. To achieve this goal, we will start by building a deep
learning framework that can analyze spatially localized RNAs in different cell types and predict their associated
regulatory factors (e.g., RNA motifs, RNA binding proteins). This will provide an atlas of spatial RNA organization
as well as candidate RNAs for functional studies. Next, we will develop two novel approaches, RNA live-cell
fluorescent in situ hybridization (RNA-LiveFISH) for single-molecule, real-time dynamic tracking, and CRISPR-
mediated transcript organization (CRISPR-TO) for programmable manipulation of any target mRNA localization.
The two approaches form a new framework that enables us to study the regulatory mechanism and functional
relevance of subcellular mRNA localization with unprecedented ease and spatiotemporal resolution. Third, we
seek to apply this framework to study the function of mRNA localization in primary neurons, via high-throughput
manipulation of >1,000 mRNAs to uncover functions for axon guidance, growth cone development, and synaptic
activities. Selected functional mRNAs (>100) will be verified in vivo. Finally, we will apply the framework to
investigate the pathological mechanisms of aberrant RNA localization underlying the neurological disease spinal
muscular atrophy (SMA) in vitro and in vivo. We will not only dissect the relationship between mRNA organization
and SMA pathology, but also explore ...

## Key facts

- **NIH application ID:** 10702050
- **Project number:** 1DP1NS137219-01
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** Lei Stanley Qi
- **Activity code:** DP1 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $1,080,800
- **Award type:** 1
- **Project period:** 2023-09-01 → 2028-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10702050

## Citation

> US National Institutes of Health, RePORTER application 10702050, Manipulating and Interrogating Spatial Transcriptomics (1DP1NS137219-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10702050. Licensed CC0.

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