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

Award Number: K99DC021984
ORGANIZATION: NATIONAL INSTITUTE ON DEAFNESS AND OTHER COMMUNICATION DISORDERS
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: TRAINING/TRAINEESHIP
PERIOD OF PERFORMANCE START DATE: 07/01/2024
PERIOD OF PERFORMANCE END DATE: 09/01/2025

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


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2026 ( Subtotal = $0 )
2026	2025	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Research Related to Deafness and Communication Disorders	000	2	12/23/2025	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2025 ( Subtotal = $21,487 )
2025	2025	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Research Related to Deafness and Communication Disorders	000	2	7/10/2025	NON-COMPETING CONTINUATION	$21,487
														Subtotal = $21,487

Issue Date FY: 2024 ( Subtotal = $128,922 )
2024	2024	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Research Related to Deafness and Communication Disorders	000	1	6/12/2024	NEW	$128,922
														Subtotal = $128,922

Grand Total All Awards = $150,409

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

Award Number: K99DC021984

ORGANIZATION: NATIONAL INSTITUTE ON DEAFNESS AND OTHER COMMUNICATION DISORDERS

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: TRAINING/TRAINEESHIP

PERIOD OF PERFORMANCE START DATE: 07/01/2024

PERIOD OF PERFORMANCE END DATE: 09/01/2025

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