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Optimizing RNA import for microinjection-free genetic manipulation in C. elegans

Award Number: R21GM159351
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 05/01/2025
PERIOD OF PERFORMANCE END DATE: 04/30/2027

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Optimizing RNA import for microinjection-free genetic manipulation in C. elegans - Project Summary C. elegans is amenable to easy genetic modifications, as proven by its pioneering and continued use of endogenously expressed fluorescent reporters and other genetically encoded molecular tools. CRISPR/Cas systems for generation of mutants and transgenic lines are widely used in C. elegans. While highly precise, CRISPR/Cas applications are severely limited by the need to microinject nucleic acids to the germline, a significantly time-consuming process that drastically hinders the use of C. elegans for high-throughput biology. On the other hand, C. elegans has the ability to uptake double stranded RNA by feeding, which has led to the generation of dsRNA-expressing bacterial libraries and their use for large-scale reverse genetic screens. In this project, we aim to address this important limitation in C. elegans by developing a strategy that enables microinjection-free CRISPR/Cas applications. We will develop an approach to efficiently deliver RNA by feeding throughout C. elegans tissues for use as single-guide RNA (sgRNA) in CRISPR/Cas systems. Our approach relies in using easily uptaken RNA carriers to facilitate efficient import of sgRNAs through ingestion. We propose to address this challenge by: 1) studying uptake of diverse RNA structures into different C. elegans tissues through the development of an RNA sensor strain, 2) designing RNA structures for optimal uptake using artificial intelligence, and 3) testing the efficacy of modified sgRNA structures in CRISPR/Cas applications. Success of this work will greatly facilitate implementation of CRISPR/Cas approaches and make large-scale high-throughput CRISPR screens possible.


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: 2025 ( Subtotal = $398,774 )
2025	2025	NORTH CAROLINA STATE UNIVERSITY	2601 WOLF VILLAGE WAY	RALEIGH	NC	27695	WAKE	USA	Biomedical Research and Research Training	000	1	4/24/2025	NEW	$398,774
														Subtotal = $398,774

Grand Total All Awards = $398,774

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Optimizing RNA import for microinjection-free genetic manipulation in C. elegans

Award Number: R21GM159351

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

PERIOD OF PERFORMANCE START DATE: 05/01/2025

PERIOD OF PERFORMANCE END DATE: 04/30/2027

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