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CRISPR-Cas13d: Transgenic zebrafish lines toknockdown mRNA

Award Number: R21OD034161
ORGANIZATION: OFFICE OF THE DIRECTOR, NIH
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 12/15/2022
PERIOD OF PERFORMANCE END DATE: 11/30/2025

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CRISPR-Cas13d: Transgenic zebrafish lines toknockdown mRNA - Project Summary Determining the function of genes is fundamental for understanding vertebrate development, regulatory mechanisms and human diseases. Genome editing technologies, such CRISPR-Cas9, have allowed associating specific phenotypes to permanent gene alteration. However, some key technical and conceptual issues remain problematic in vertebrates, particularly in aquatic model organisms such zebrafish. For example, the maternally provided mRNA can rescue the phenotype of homozygous mutants; genotyping steps are tedious; and long non- coding RNA, lethal or tissue/temporal genes are difficult to study using DNA manipulation as well as complex genomic loci. Complementary ‘knock-down’ approaches are invaluable tools to circumvent some of these problems, however, there was no systematic tool to knockdown mRNAs in zebrafish or other teleost fish. Our long-term goal is to unravel the function of genes related to regulatory mechanisms, development, and human diseases. Our recent publication demonstrates that injection of the CRISPR-RfxCas13d system into vertebrate embryos provides a robust and cost-effective technology to systematically disrupt gene function. However, the injection of this system only provides transient knockdown for ~3 days. Therefore, the central goal of the proposal is to transfer the CRISPR-RfxCas13d technology from being injected into vertebrate embryos, to being endogenously expressed through transgenesis. We propose that the development of a repertoire transgenic zebrafish expressing the RfxCas13d enzyme (ubiquitous and tissues-specific) and guideRNA would facilitate rapid and vigorous investigation into gene functions. Our preliminary data indicate that transgenic expression of RfxCas13d in zebrafish is functional and not toxic. The objectives are: 1) Define the optimal system to knockdown mRNA expression in CRISPR-RfxCas13d transgenic zebrafish. 2) Knockdown mRNA expression in a tissue-specific manner using specific transgenic lines. This proposal is conceptually innovative as it is based on the exploration of a novel technique, CRISPR-RfxCas13d, to knockdown mRNA in a tissue-specific manner in zebrafish. This approach has never been done in vertebrate model systems where RNAi does not work. The outcomes of this project will be the first transgenic system to dissect and study gene function by knocking down mRNA expression in zebrafish. Our approach will help the scientific community investigate gene function in a faster and tissue-temporal specific manner, as well as answering questions that are very challenging to address by current methodologies, such as the function of non-coding RNAs or phenotypes caused by multiple genes. Moreover, by following the guidelines we will optimize through this project, researchers may produce a whole guideRNA collection, targeting all genes, which should be available for anyone to order to address their individual research questions. Finally, as we have successfully implemented the CRISPR-Cas13d system by injection in other organisms such as medaka, killifish, and mouse embryos, our work described in this proposal may serve as the foundation for transferring this efficient knockdown technology into a range of other species.


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	2024	STOWERS INSTITUTE FOR MEDICAL RESEARCH	1000 E 50TH ST	KANSAS CITY	MO	64110	JACKSON	USA	Research Infrastructure Programs	000	2	3/3/2026	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2025 ( Subtotal = $0 )
2025	2024	STOWERS INSTITUTE FOR MEDICAL RESEARCH	1000 E 50TH ST	KANSAS CITY	MO	64110	JACKSON	USA	Research Infrastructure Programs	000	2	11/1/2024	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2024 ( Subtotal = $247,500 )
2024	2024	STOWERS INSTITUTE FOR MEDICAL RESEARCH	1000 E 50TH ST	KANSAS CITY	MO	64110	JACKSON	USA	Research Infrastructure Programs	001	2	5/10/2024	NON-COMPETING CONTINUATION	$49,500
2024	2024	STOWERS INSTITUTE FOR MEDICAL RESEARCH	1000 E 50TH ST	KANSAS CITY	MO	64110	JACKSON	USA	Research Infrastructure Programs	000	2	11/8/2023	NON-COMPETING CONTINUATION	$198,000
														Subtotal = $247,500

Issue Date FY: 2023 ( Subtotal = $206,250 )
2023	2023	STOWERS INSTITUTE FOR MEDICAL RESEARCH	1000 E 50TH ST	KANSAS CITY	MO	64110	JACKSON	USA	Research Infrastructure Programs	000	1	12/9/2022	NEW	$185,625
2023	2023	STOWERS INSTITUTE FOR MEDICAL RESEARCH	1000 E 50TH ST	KANSAS CITY	MO	64110	JACKSON	USA	Research Infrastructure Programs	001	1	2/22/2023	NEW	$20,625
2023	2023	STOWERS INSTITUTE FOR MEDICAL RESEARCH	1000 E 50TH ST	KANSAS CITY	MO	64110	JACKSON	USA	Research Infrastructure Programs	002	1	7/19/2023	NEW	$0
														Subtotal = $206,250

Grand Total All Awards = $453,750

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CRISPR-Cas13d: Transgenic zebrafish lines toknockdown mRNA

Award Number: R21OD034161

ORGANIZATION: OFFICE OF THE DIRECTOR, NIH

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 12/15/2022

PERIOD OF PERFORMANCE END DATE: 11/30/2025

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