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Characterization of structural elements controlling Cas9-mediated DNA cleavage and single-turnover enzyme kinetics.

Award Number: F31GM143822
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS

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Characterization of structural elements controlling Cas9-mediated DNA cleavage and single-turnover enzyme kinetics. - ABSTRACT The CRISPR/Cas9 system offers an extremely versatile genome editing technology by employing an RNA- guided endonuclease, Cas9. Originally isolated from Streptococcus pyogenes, this system has been adapted for use in a wide range of organisms and can be programmed to manipulate almost any site in the genome. While Cas9 is easy to reprogram, the efficiency of Cas9-mediated editing varies considerably across different genomic targets. Unlike other common bacterial endonucleases, Cas9 exhibits single-turnover kinetics where it forms a stable product complex and requires an external force to release the cut DNA. This persistent product state impairs access to the double-strand break by repair machinery and contributes to reduced genome editing efficiency. Despite an abundance of Cas9 structures, the structure of the product complex remains uncharacterized as previous studies were carried out under conditions that prevent DNA cleavage. As a result, our structural understanding of the entire Cas9 reaction cycle remains incomplete and insufficient to explain why Cas9 exhibits single-turnover kinetics. Our lab recently used cryo-EM to determine the structure of the catalytically active Cas9-sgRNA-dsDNA ternary complex and captured three distinct conformational states (pre- and post-catalytic, and product states). These structures provide new insight into the coordination of Cas9 domains throughout catalysis and reveal persistent Cas9-nucleic acid interactions in the product state. Guided by this new insight, we will expand upon these studies to define the major structural elements contributing to Cas9 catalysis and single-turnover kinetics. Using innovative in vitro kinetic assays and established structural analyses, we will investigate the structural features that control the rate at which Cas9 cuts and releases the targeted DNA. Using rational design in conjunction with random mutagenesis, we will engineer a multi-turnover enzyme capable of spontaneously releasing its cleaved DNA product. Using an in vitro genome editing assay and an in vivo -galactosidase assay, we will screen Cas9 mutants for an increased rate of DNA cleavage. We anticipate the proposed studies will not only advance our molecular understanding of the Cas9 reaction cycle, but also support the development of more efficient CRISPR/Cas9 technologies.


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 = -$16,308 )
2024	2023	UNIVERSITY OF ILLINOIS	809 S MARSHFIELD AVE M/C 551	CHICAGO	IL	60612	COOK	USA	Biomedical Research and Research Training	000	1	2/6/2024	NEW	-$13,269
2024	2023	UNIVERSITY OF ILLINOIS	809 S MARSHFIELD AVE M/C 551	CHICAGO	IL	60612	COOK	USA	Biomedical Research and Research Training	001	1	9/23/2024	NEW	-$3,039
														Subtotal = -$16,308

Issue Date FY: 2023 ( Subtotal = $44,094 )
2023	2023	UNIVERSITY OF ILLINOIS	809 S MARSHFIELD AVE M/C 551	CHICAGO	IL	60612	COOK	USA	Biomedical Research and Research Training	001	1	1/1/2023	NEW	$0
2023	2023	UNIVERSITY OF ILLINOIS	809 S MARSHFIELD RM 520	CHICAGO	IL	60612	COOK	USA	Biomedical Research and Research Training	002	1	3/1/2023	NEW	$942
2023	2023	UNIVERSITY OF ILLINOIS	809 S MARSHFIELD AVE M/C 551	CHICAGO	IL	60612	COOK	USA	Biomedical Research and Research Training	003	1	5/15/2023	NEW	$0
2023	2023	UNIVERSITY OF ILLINOIS	809 S MARSHFIELD RM 520	CHICAGO	IL	60612	COOK	USA	Biomedical Research and Research Training	000	1	1/1/2023	NEW	$43,152
														Subtotal = $44,094

Grand Total All Awards = $27,786

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Characterization of structural elements controlling Cas9-mediated DNA cleavage and single-turnover enzyme kinetics.

Award Number: F31GM143822

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS

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