Thursday, November 21, 2024 11/21/2024

Molecular mechanisms of RNA-targeting CRISPR-Cas systems and their membrane-associated accessory proteins

Award Number: R35GM133462
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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Microbes employ diverse defense systems to provide protection from viruses, and studying these systems is important for understanding immune system evolution, phage therapy, and the discovery of new molecular tools such as CRISPR-Cas systems. While it’s broadly thought that CRISPR-Cas systems work exclusively by degrading nucleic acids, we’ve recently shown that membrane perturbation by accessory proteins (e.g., Csx28) are an unappreciated aspect of CRISPR-Cas’s mechanism of action. However, the molecular mechanisms of how proteins like Csx28 function are unknown. The goal of my research is to understand how CRISPR-Cas systems and their membrane-associated proteins function together to enhance anti-viral defense with an eye towards the discovery of new tools. This proposal focuses on addressing the following gaps: 1) What is the mechanistic and structural details of Csx28 activation by Cas13? We recently discovered that the RNA-targeting Cas enzyme Cas13 is communicating with membrane pore protein Csx28, to mount a robust anti-viral immune response, however we still don’t understand what the precise molecular mechanism involved. Our preliminary data indicate that Cas13 generated RNA cleavage products are acting as ligands to gate Csx28 channel activity. We will use RNA sequencing and membrane biophysics approaches to determine the precise RNA ligands modulating Csx28 activity and how these ligands affect gating of the Csx28 pore, respectively. These experiments will shed light on the molecular details of fascinating anti-phage synergy between Cas13 and Csx28 and provide a clear path for technology development. 2) Do Csx28-like proteins have roles in anti-viral defense outside of CRISPR systems? With our structural studies of Csx28 coupled with the recent advances in structure prediction databases, we discovered that Csx28 shares very close structural similarity to a family of hypothetical proteins that reside in potentially new immune systems. We hypothesize that these Csx28-like proteins are employed to modulate membrane function during viral infection, however no experimental studies have been done. We will use similar genetic, biochemical, and structural approaches to our previous work on Csx28 to study these putative membrane proteins. 3) How does Cas13 associated protein Csx27 modulate antiphage defense? Csx27 is a predicted transmembrane CRISPR accessory gene that resides within a subset of Cas13b containing loci. Csx27 has recently been shown to work with Cas13b from to modulate anti-phage defense, however the mechanism is not understood. Our preliminary data suggest that Csx27, while bearing no similarity to Csx28, also acts as a membrane pore and activation by Cas13b results in a similar loss of membrane potential, and a muted metabolism, however more work needs to be done to understand the details these of effects. We will use a similar set of genetic, biochemical, and structural approaches that were successful previously for Csx28 with the goal of understanding of Csx27’s role in Cas13b antiphage defense, and to pave the way for downstream technology e.g., potential applications that tie RNA expression levels to the modulation of membrane behavior.


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	2024	UNIVERSITY OF ROCHESTER	910 GENESEE ST	ROCHESTER	NY	14611	MONROE	USA	Biomedical Research and Research Training	000	6	11/6/2024	COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2024 ( Subtotal = $452,437 )
2024	2024	UNIVERSITY OF ROCHESTER	910 GENESEE ST	ROCHESTER	NY	14611	MONROE	USA	Biomedical Research and Research Training	000	6	3/26/2024	COMPETING CONTINUATION	$452,437
														Subtotal = $452,437

Issue Date FY: 2023 ( Subtotal = $385,000 )
2023	2023	UNIVERSITY OF ROCHESTER	910 GENESEE ST	ROCHESTER	NY	14611	MONROE	USA	Biomedical Research and Research Training	000	5	5/22/2023	NON-COMPETING CONTINUATION	$385,000
														Subtotal = $385,000

Issue Date FY: 2022 ( Subtotal = $530,283 )
2022	2022	UNIVERSITY OF ROCHESTER	500 JOSEPH C WILSON BLVD	ROCHESTER	NY	14627	MONROE	USA	Biomedical Research and Research Training	000	4	5/13/2022	NON-COMPETING CONTINUATION	$385,000
2022	2022	UNIVERSITY OF ROCHESTER	500 JOSEPH C WILSON BLVD	ROCHESTER	NY	14627	MONROE	USA	Biomedical Research and Research Training	001	4	6/10/2022	SUPPLEMENT FOR EXPANSION	$145,283
														Subtotal = $530,283

Issue Date FY: 2021 ( Subtotal = $480,644 )
2021	2021	UNIVERSITY OF ROCHESTER	500 JOSEPH C WILSON BLVD	ROCHESTER	NY	14627	MONROE	USA	Biomedical Research and Research Training	000	3	5/18/2021	NON-COMPETING CONTINUATION	$385,000
2021	2021	UNIVERSITY OF ROCHESTER	500 JOSEPH C WILSON BLVD	ROCHESTER	NY	14627	MONROE	USA	Biomedical Research and Research Training	001	3	8/3/2021	SUPPLEMENT FOR EXPANSION	$95,644
														Subtotal = $480,644

Issue Date FY: 2020 ( Subtotal = $431,902 )
2020	2020	UNIVERSITY OF ROCHESTER	500 JOSEPH C WILSON BLVD	ROCHESTER	NY	14627	MONROE	USA	Biomedical Research and Research Training	001	2	7/8/2020	SUPPLEMENT FOR EXPANSION	$46,902
2020	2020	UNIVERSITY OF ROCHESTER	500 JOSEPH C WILSON BLVD	ROCHESTER	NY	14627	MONROE	USA	Biomedical Research and Research Training	000	2	5/7/2020	NON-COMPETING CONTINUATION	$385,000
														Subtotal = $431,902

Issue Date FY: 2019 ( Subtotal = $385,000 )
2019	2019	UNIVERSITY OF ROCHESTER	500 JOSEPH C WILSON BLVD	ROCHESTER	NY	14627	MONROE	USA	Biomedical Research and Research Training	000	1	7/26/2019	NEW	$385,000
														Subtotal = $385,000

Grand Total All Awards = $2,665,266

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Molecular mechanisms of RNA-targeting CRISPR-Cas systems and their membrane-associated accessory proteins

Award Number: R35GM133462

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

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