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CRISPR-based editing of RNA and cellular mechanisms that govern RNA repair

Award Number: K99AI171893
ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: TRAINING/TRAINEESHIP
PERIOD OF PERFORMANCE START DATE: 08/17/2022
PERIOD OF PERFORMANCE END DATE: 07/31/2024

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CRISPR-based editing of RNA and cellular mechanisms that govern RNA repair - PROJECT SUMMARY CRISPR-based technologies have transformed science by enabling targeted DNA modification. DNA editing technologies have enriched our mechanistic understanding of DNA repair, and new insights in repair are frequently used to improve methods for gene editing. However, despite the remarkable achievements in targeted DNA modifications, RNA repair is understudied, and RNA editing tools are limited. The long-term goal of this proposal is to understand the molecular mechanisms that govern RNA repair and to develop disruptive new RNA editing tools for applications in science and medicine. The research proposed here integrates synergistic efforts to develop CRISPR-based tools for dissecting viral mechanisms of host cell take over and identify viral RNA repair mechanisms that support acute respiratory syndrome coronavirus (SARS-CoV-2) replication. In Aim 1, I will repurpose RNA-targeting CRISPR systems for deleting, inserting, and substituting sequences in viral RNAs. I have designed CRISPR-Cas systems to delete regions of ORF7a in the SARS- CoV-2 genome and recreate naturally occurring mutations. Based on my previous work, I anticipate that targeted deletions in ORF7a limit viral suppression of the host interferon response and lead to a replication defect. Further, tools developed in this application will be used to test emerging viral variants for new phenotypes. Aim 2 investigates the antagonistic activities of host antiviral nucleases and host RNA ligases in the replication and evolution of SARS-CoV-2. Aim 3 develops a CRISPR-based RNA capture system to enrich sequence-specific RNAs from a complex mixture. I will use CRISPR-based enrichment to map RNA modifications in SARS-CoV-2 viral RNAs and identify molecular signatures associated with interspecies transitions. Successful completion of the K99 phase of this application will require training in the Biosafety Level 3 facility, bioinformatics training to identify and annotate CRISPR systems, as well as new competencies in RNA biochemistry. Collectively, the research and training objectives outlined here establish a solid scientific foundation that will facilitate my transition to independence.


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 = $0 )
2025	2023	MONTANA STATE UNIVERSITY	216 MONTANA HALL	BOZEMAN	MT	59717	GALLATIN	USA	Allergy and Infectious Diseases Research	000	2	4/10/2025	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2023 ( Subtotal = $137,700 )
2023	2023	MONTANA STATE UNIVERSITY	216 MONTANA HALL	BOZEMAN	MT	59717	GALLATIN	USA	Allergy and Infectious Diseases Research	000	2	7/12/2023	NON-COMPETING CONTINUATION	$137,700
														Subtotal = $137,700

Issue Date FY: 2022 ( Subtotal = $138,240 )
2022	2022	MONTANA STATE UNIVERSITY	216 MONTANA HALL	BOZEMAN	MT	59717	GALLATIN	USA	Allergy and Infectious Diseases Research	000	1	8/17/2022	NEW	$138,240
														Subtotal = $138,240

Grand Total All Awards = $275,940

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CRISPR-based editing of RNA and cellular mechanisms that govern RNA repair

Award Number: K99AI171893

ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: TRAINING/TRAINEESHIP

PERIOD OF PERFORMANCE START DATE: 08/17/2022

PERIOD OF PERFORMANCE END DATE: 07/31/2024

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