In Vivo Targeting of Clostridioides difficile Using Phage-Delivered CRISPR-Cas3 Antimicrobials.

MBio
Kurt SelleDavid G Ousterout

Abstract

Clostridioides difficile is an important nosocomial pathogen that causes approximately 500,000 cases of C. difficile infection (CDI) and 29,000 deaths annually in the United States. Antibiotic use is a major risk factor for CDI because broad-spectrum antimicrobials disrupt the indigenous gut microbiota, decreasing colonization resistance against C. difficile Vancomycin is the standard of care for the treatment of CDI, likely contributing to the high recurrence rates due to the continued disruption of the gut microbiota. Thus, there is an urgent need for the development of novel therapeutics that can prevent and treat CDI and precisely target the pathogen without disrupting the gut microbiota. Here, we show that the endogenous type I-B CRISPR-Cas system in C. difficile can be repurposed as an antimicrobial agent by the expression of a self-targeting CRISPR that redirects endogenous CRISPR-Cas3 activity against the bacterial chromosome. We demonstrate that a recombinant bacteriophage expressing bacterial genome-targeting CRISPR RNAs is significantly more effective than its wild-type parent bacteriophage at killing C. difficile both in vitro and in a mouse model of CDI. We also report that conversion of the phage from temperate to...Continue Reading

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Citations

Jan 17, 2021·Journal of Periodontal Research·Abdelahhad BarbourMichael Glogauer
Dec 15, 2020·Current Opinion in Biotechnology·Bryan R LennemanSamuel Kilcher
Dec 29, 2020·Current Opinion in Biotechnology·Clément FageSylvain Moineau
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Aug 28, 2021·Antibiotics·Aa Haeruman AzamLongzhu Cui
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Methods Mentioned

BETA
genetic
transmission electron microscopy
gene knockout
PCR
electrophoresis

Software Mentioned

GraphPad
cellSens Dimension
Gatan Microscopy (
Prism

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