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Targeted ingression of SNPs into the zebrafish genome

Award Number: R21OD036481
ORGANIZATION: OFFICE OF THE DIRECTOR, NIH
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 09/15/2024
PERIOD OF PERFORMANCE END DATE: 08/31/2027

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Targeted ingression of SNPs into the zebrafish genome - Despite recent advances in gene editing, the ability to make small changes in the genomes of model organisms remains a challenge. In zebrafish, enhanced oligo design, base-editing and prime-editing show promise to increase the frequencies of small changes but their application is still cumbersome due to low germline transmission rates, requiring extensive and time-consuming screening. This bottleneck prevents the efficient targeted production single nucleotide polymorphisms (SNPs) for disease modeling, limits our ability to perform mechanistic structure-function studies, and prevents rigorous determination and the assessment of variants of uncertain significance (VUS) that promote human disease. The overarching goal of the proposed experiments is to create a gene editing method to efficiently introduce SNPs and VAPs into the zebrafish genome for modeling human health. We have used short regions of homology surrounding a CRISPR/Cas9 double strand breaks to efficiently target integration of DNA cargos for mutagenesis, protein tagging, Gal4 reporters, Cre-drivers and conditional alleles, using a method we have coined GeneWeld. Key to this methodology is repair at a double strand break in genome produced by injection of Cas9 mRNA and a genomic gRNA with a plasmid template. The circular plasmid template is cut in vivo with a gRNA, called a universal gRNA (3), that recognizes sequences adjacent to the short homology to produce a linear template for integration. The repair and subsequent integration of the cargo is likely utilizing microhomology-mediated end joining (MMEJ) or a single-strand annealing (SSA) mechanism and results in germline transmission of the targeted integration events in approximately 50% of the F0 injected embryos to the next generation. Since this methodology remains more efficient that other strategies for targeted CRISPR/Cas9-targeted integration in zebrafish, we propose that GeneWeld can be used to efficiently target ingression of SNPs and VUS into the zebrafish genome. For this, we will test the ability of GeneWeld to introduce SNPs at three different loci. We will also examine whether co-selection of RFP repair correlates with ingression of small changes in the genome at a separate gene. Additionally, we will enhance the ability to generate SNPs and VUS in zebrafish by overexpression of DNA repair enzymes and dominant mutations in repair enzymes. Key preliminary data indicates that overexpression of a variant of Rad51 reproducibly enhances repair. We will use this approach to examine the activity of Rad51 for the ingression of small nucleotide changes. The proposed experiments are expected to develop simple and efficient methods to target SNPs and VUS into any gene in the zebrafish genome. In addition, we will generate an important model with a dead RFP knock-in that will be of great utility to assess editing and may provide a means for co-selection of editing events. Based on the ability of GeneWeld to be used in other organisms, we expect these methodologies to be broadly applicable to animal models used by the NIH.


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	IOWA STATE UNIVERSITY OF SCIENCE AND TECHNOLOGY	1350 BEARDSHEAR HALL	AMES	IA	50011	STORY	USA	Research Infrastructure Programs	000	2	6/5/2026	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2025 ( Subtotal = $219,605 )
2025	2025	IOWA STATE UNIVERSITY OF SCIENCE AND TECHNOLOGY	1350 BEARDSHEAR HALL	AMES	IA	50011	STORY	USA	Research Infrastructure Programs	000	2	8/20/2025	NON-COMPETING CONTINUATION	$219,605
														Subtotal = $219,605

Issue Date FY: 2024 ( Subtotal = $181,754 )
2024	2024	IOWA STATE UNIVERSITY OF SCIENCE AND TECHNOLOGY	1350 BEARDSHEAR HALL	AMES	IA	50011	STORY	USA	Research Infrastructure Programs	000	1	9/3/2024	NEW	$181,754
														Subtotal = $181,754

Grand Total All Awards = $401,359

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Targeted ingression of SNPs into the zebrafish genome

Award Number: R21OD036481

ORGANIZATION: OFFICE OF THE DIRECTOR, NIH

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

PERIOD OF PERFORMANCE START DATE: 09/15/2024

PERIOD OF PERFORMANCE END DATE: 08/31/2027

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