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Viral vector-mediated gene activation to facilitate large-scale genetic analysis in Caenorhabditis elegans.

Award Number: R21GM148859
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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Viral vector-mediated gene activation to facilitate large-scale genetic analysis in Caenorhabditis elegans. - Project Summary Fulfilling the promise of modern systems biology and grasping the underlying complexity of biological systems requires a foundation built upon the development of high-throughput functional genomic approaches capable of generating large datasets. Large scale sequencing efforts reveal correlations, but lacks causal interactions best provided via genetic approaches. Caenorhabditis elegans has been a workhorse for gene discovery and pathway analysis, and is the only established system where high-throughput genetic analysis can be conducted in the context of a living multi-cellular organism (i.e. feeding based RNAi). Despite the power of this model system, no high-throughput methods to achieve targeted gene overexpression in C. elegans have been developed. This project will explore how recombinant strains of two different viruses can be adapted as vectors to enable large-scale genetic analysis of gene overexpression in C. elegans. The objective of Specific Aim 1 is to achieve promoter-specific gene activation using CRISPRa. This variant form of CRISPR relies on a cleavage defective isoform of Cas9 (dCas9) fused with a transcriptional activator to drive overexpression of a gene targeted by the single gene RNA (sgRNA). Specifically, we propose to generate proof-of-principle evidence that recombinant vesicular stomatitis virus (rVSV) can deliver a sgRNA into transgenic C. elegans that express the CRISPRa machinery in intestinal cells to induce sgRNA-directed overexpression of a reporter gene. Ultimately our goal is to develop a comprehensive sgRNA VSV library directed to promoter regions to allow high-throughput functional genomic screening in C. elegans. The objective of Specific Aim 2 is to develop Orsay virus (OV) as a vector to deliver functional mRNA exogenously into C. elegans. The use of OV as a gene delivery system is straightforward as this virus readily enters the animal via the intestinal lumen, and C. elegans expressing integrated segments of the OV genome have been validated. Briefly, we will use these existing strains as “packaging lines” to express C. elegans genes of interest capable of being incorporated in newly generated virion to infect recipient nematodes. These studies represent an initial step towards the use of OV as an overexpression vector and would accelerate the development large-scale genetic analysis in this multicellular organism. These viral-based expression tools would integrate easily with existing approaches widely used by the C. elegans community, which could potentially transform multiple areas of scientific investigation, and has implications for understanding of many diseases.


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: 2024 ( Subtotal = $216,625 )
2024	2024	UNIVERSITY OF ROCHESTER	910 GENESEE ST	ROCHESTER	NY	14611	MONROE	USA	Biomedical Research and Research Training	001	2	1/19/2024	SUPPLEMENT FOR EXPANSION	$31,005
2024	2024	UNIVERSITY OF ROCHESTER	910 GENESEE ST	ROCHESTER	NY	14611	MONROE	USA	Biomedical Research and Research Training	002	2	6/18/2024	NON-COMPETING CONTINUATION	$18,562
2024	2024	UNIVERSITY OF ROCHESTER	910 GENESEE ST	ROCHESTER	NY	14611	MONROE	USA	Biomedical Research and Research Training	000	2	1/18/2024	NON-COMPETING CONTINUATION	$167,058
														Subtotal = $216,625

Issue Date FY: 2023 ( Subtotal = $224,280 )
2023	2023	UNIVERSITY OF ROCHESTER	500 JOSEPH C WILSON BLVD	ROCHESTER	NY	14627	MONROE	USA	Biomedical Research and Research Training	000	1	1/12/2023	NEW	$205,192
2023	2023	UNIVERSITY OF ROCHESTER	910 GENESEE ST	ROCHESTER	NY	14611	MONROE	USA	Biomedical Research and Research Training	001	1	9/18/2023	SUPPLEMENT FOR EXPANSION	$19,088
														Subtotal = $224,280

Grand Total All Awards = $440,905

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Viral vector-mediated gene activation to facilitate large-scale genetic analysis in Caenorhabditis elegans.

Award Number: R21GM148859

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

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