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CRISPR interference-enabled phenotyping of essential genes in C. difficile to aid in discovery of antibiotic targets

Award Number: R21AI159071
ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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Project Summary Clostridioides (Clostridium) difficile infections strike close to 500,000 people a year in the United States, leading to nearly 30,000 deaths. The CDC has declared this organism an “urgent” threat to public health, the highest threat category. New treatments are sorely needed. Most antibiotics inhibit proteins that are essential for viability, so a better understanding of these proteins and the genes that encode them can advance efforts to develop new antibiotics. Although essential genes are difficult to study, new techniques for manipulating bacterial gene expression are overcoming this barrier. One emerging technology for studying essential genes in bacteria is called CRISPR interference (CRISPRi). CRISPRi uses a short RNA to guide a protein called dCas9 to a gene, thereby creating a roadblock that prevents RNA polymerase from transcribing that gene. CRISPRi is well-suited for screening large sets of genes because repressing a new target is simply a matter of cloning a synthetic DNA sequence encoding the right guide RNA into the plasmid, a process that is easily scaled-up. Another key feature of our CRISPRi system is that dCas9 is expressed from a xylose-inducible promoter, which allows us to control the timing and extent of gene repression. The specific objective of this proposal is to deploy CRISPRi to characterize 187 putatively essential genes in C. difficile. In Aim 1 we will construct a CRISPRi library and use it to vet the essentiality of the genes on our target list. In Aim 2 we will determine whether gene knockdown results in morphological defects or increases sensitivity to a panel of antibiotics chosen to target a variety of cellular processes. The information from these assays will enable us to assign many of these genes to functional pathways. It will also provide foundational knowledge and tools for developing new antibiotics to treat C. difficile infections.


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: 2023 ( Subtotal = $173,813 )
2023	2023	UNIVERSITY OF IOWA, THE	105 JESSUP HALL	IOWA CITY	IA	52242	JOHNSON	USA	Allergy and Infectious Diseases Research	000	2	10/28/2022	NON-COMPETING CONTINUATION	$173,813
														Subtotal = $173,813

Issue Date FY: 2022 ( Subtotal = $208,575 )
2022	2022	UNIVERSITY OF IOWA, THE	105 JESSUP HALL	IOWA CITY	IA	52242	JOHNSON	USA	Allergy and Infectious Diseases Research	000	1	11/2/2021	NEW	$208,575
														Subtotal = $208,575

Grand Total All Awards = $382,388

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CRISPR interference-enabled phenotyping of essential genes in C. difficile to aid in discovery of antibiotic targets

Award Number: R21AI159071

ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

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