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Nucleic Acid-Based Anti-CRISPR Inhibitors of Cas9

Award Number: R01GM135646
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: 07/05/2023
PERIOD OF PERFORMANCE END DATE: 12/31/2024

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Nucleic Acid-Based Anti-CRISPR Inhibitors of Cas9 - PROJECT SUMMARY Sophisticated, facile, and potent inhibitors of Cas enzymes that are simple to use are still a major missing tool for CRISPR-based biomedical and therapeutic research. The rationale for CRISPR-Cas inhibitors includes a need for more efficient generation of model cells and organisms, the threat of dangerous off-target editing in CRISPR-based therapeutics, and the desire for more precise control over Cas enzyme activity in synthetic biology and diagnostics applications. Methods and modified Cas enzymes have been developed to help address these issues and other shortcomings of CRISPR-Cas systems, particularly Cas9 from Streptococcus pyogenes (SpCas9). These include Cas9 mutants with lower off-target activity and Cas9 mutants and protein fusions that allow small molecule or light-based activation of Cas9. However, these modifications typically have a concomitant decrease in on-target activity and do not address the need for rapid and specific shut-down of Cas9. Despite improvements to Cas enzymes, the need for specific, broadly applicable, and easy-to-use inhibitors will remain a necessary tool that is not available. Small molecules that can inhibit Cas9 may take significant effort to develop and potentially come with downsides, including their own off-target effects and inadvertent impacts on the organism’s microbiota, possibly similar to antibiotic side-effects. Natural anti-CRISPR proteins have recently been discovered that bind Cas9 and other Cas proteins with high affinity. While promising as inhibitors, they are too large to possess drug-like properties and their minimization or optimization is not an obvious exercise. They must be genetically encoded for use in cells or organisms. However, they provide inspiration for the design of a new class of CRISPR-Cas inhibitors. Here we propose to develop a new technology, nucleic acid-based (NAB) inhibitors of Cas9 enzymes. These molecules are smaller than natural anti-CRISPR proteins but can bind with similar affinity, be chemically synthesized, and be readily introduced into cells with common methods. In this project, we will develop NAB inhibitor technology by optimizing their size, chemistry, binding affinity, inhibitory activity, and cellular stability. We will further develop methods and molecules that facilitate direct, carrier-free delivery and timed-release of NAB inhibitors. The resulting NAB inhibitors are expected to be broadly applicable and straightforward to use for diverse biomedical research. We expect NAB inhibitors to become valuable fail-safe inhibitors to overcome the critical safety hurdles in CRISPR-based therapeutics.


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 = -$145 )
2025	2023	WAKE FOREST UNIVERSITY HEALTH SCIENCES	MEDICAL CENTER BLVD	WINSTON SALEM	NC	27157	FORSYTH	USA	Biomedical Research and Research Training	000	5	5/6/2025	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	-$145
														Subtotal = -$145

Issue Date FY: 2024 ( Subtotal = $0 )
2024	2023	BOARD OF TRUSTEES OF SOUTHERN ILLINOIS UNIVERSITY	900 S NORMAL AVE	CARBONDALE	IL	62901	JACKSON	USA	Biomedical Research and Research Training	000	4	6/7/2024	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2023 ( Subtotal = $294,698 )
2023	2023	SOUTHERN ILLINOIS UNIVERSITY	900 SOUTH NORMAL AVE	CARBONDALE	IL	62901	JACKSON	USA	Biomedical Research and Research Training	000	4	12/21/2022	NON-COMPETING CONTINUATION	$242,748
2023	2023	SOUTHERN ILLINOIS UNIVERSITY	900 SOUTH NORMAL AVE	CARBONDALE	IL	62901	JACKSON	USA	Biomedical Research and Research Training	001	4	2/16/2023	NON-COMPETING CONTINUATION	$26,972
2023	2023	SOUTHERN ILLINOIS UNIVERSITY	900 SOUTH NORMAL AVE	CARBONDALE	IL	62901	JACKSON	USA	Biomedical Research and Research Training	002	4	8/3/2023	NON-COMPETING CONTINUATION	-$216,948
2023	2023	WAKE FOREST UNIVERSITY HEALTH SCIENCES	MEDICAL CENTER BLVD	WINSTON SALEM	NC	27157	FORSYTH	USA	Biomedical Research and Research Training	003	5	8/4/2023	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$241,926
														Subtotal = $294,698

Issue Date FY: 2022 ( Subtotal = $269,720 )
2022	2022	SOUTHERN ILLINOIS UNIVERSITY	900 SOUTH NORMAL AVE	CARBONDALE	IL	62901	JACKSON	USA	Biomedical Research and Research Training	000	3	1/14/2022	NON-COMPETING CONTINUATION	$242,748
2022	2022	SOUTHERN ILLINOIS UNIVERSITY	900 SOUTH NORMAL AVE	CARBONDALE	IL	62901	JACKSON	USA	Biomedical Research and Research Training	001	3	6/22/2022	NON-COMPETING CONTINUATION	$26,972
														Subtotal = $269,720

Issue Date FY: 2021 ( Subtotal = $269,720 )
2021	2021	SOUTHERN ILLINOIS UNIVERSITY	900 SOUTH NORMAL AVE	CARBONDALE	IL	62901	JACKSON	USA	Biomedical Research and Research Training	000	2	12/28/2020	NON-COMPETING CONTINUATION	$242,748
2021	2021	SOUTHERN ILLINOIS UNIVERSITY	900 SOUTH NORMAL AVE	CARBONDALE	IL	62901	JACKSON	USA	Biomedical Research and Research Training	001	2	2/17/2021	NON-COMPETING CONTINUATION	$26,972
														Subtotal = $269,720

Issue Date FY: 2020 ( Subtotal = $281,595 )
2020	2020	SOUTHERN ILLINOIS UNIVERSITY	900 SOUTH NORMAL AVE	CARBONDALE	IL	62901	JACKSON	USA	Biomedical Research and Research Training	000	1	1/3/2020	NEW	$281,595
														Subtotal = $281,595

Grand Total All Awards = $1,115,588

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Nucleic Acid-Based Anti-CRISPR Inhibitors of Cas9

Award Number: R01GM135646

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: 07/05/2023

PERIOD OF PERFORMANCE END DATE: 12/31/2024

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