# A CRISPR-based toolkit for investigating hair cell transcription factors in inner ear organoids

> **NIH NIH K99** · STANFORD UNIVERSITY · 2024 · $128,922

## Abstract

PROJECT SUMMARY/ABSTRACT
Hair cells (HCs) are the specialized sensory cells of the inner ear that play a crucial role in transmitting
environmental information, such as sound and motion, to the brain. In adult mammals, HCs cannot regenerate
naturally. Therefore, a large effort has been made to understand the normal development of auditory and
vestibular HCs, so that effective regenerative therapies can be established. Technological advancements in
studying transcriptional control have provided valuable insights into the roles of different transcription factors
(TFs) such as ATOH1, POU4F3, GFI1, INSM1, TBX2, and IKZF2 in the development of HCs. However, the
application of certain tools, such as CRISPR-mediated gene activation and inhibition, for studying HC
development has been limited by the complicated use of animal models. Recently, a highly scalable protocol
was developed to guide mouse embryonic stem cells (mESCs) towards inner ear sensory cells in vitro. Over a
span of 2-3 weeks, these inner ear organoids produce sensory epithelia-like structures complete with
mechanosensitive HCs and underlying supporting cells. The development of this inner ear organoid model
facilitates the progress of cell-based assays for studying HC-specific development and testing potential
treatments for sensory cell loss. In this proposal, I aim to utilize the inner ear organoid model to investigate the
transcriptional control of HC development in vitro. To achieve this, I plan to:
I. Define the TFs involved in in vitro inner ear organoid HC development compared to in vivo HC
development.
II. Generate a versatile toolkit of mESC lines designed for inducible and reversible CRISPR-mediated gene
 activation and inhibition.
III. Utilize this toolkit to systematically investigate the roles of candidate TFs in organoid HCs through
 CRISPR-mediated knockdown and activation, followed by multi-omic analyses.
Successful completion of this project will not only uncover additional TFs essential for HC development but will
also provide a valuable resource for fellow inner ear researchers seeking to manipulate the expression of genes
of interest within inner ear organoid cell types.

## Key facts

- **NIH application ID:** 10947953
- **Project number:** 1K99DC021984-01
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** Maggie S. Matern
- **Activity code:** K99 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $128,922
- **Award type:** 1
- **Project period:** 2024-07-01 → 2026-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10947953

## Citation

> US National Institutes of Health, RePORTER application 10947953, A CRISPR-based toolkit for investigating hair cell transcription factors in inner ear organoids (1K99DC021984-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10947953. Licensed CC0.

---

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