Research at North Carolina State University shows that the CRISPR-Cas system can be used to effectively target and eliminate specific gut bacteria, in this case Clostridioides difficile, the pathogen that causes colitis – a chronic, degenerative disease of the colon.

In a proof-of-concept study published in the journal mBio, researchers were able to show pathogen reductions in experiments conducted both on the lab bench and in mice.

Microbiologists from two different NC State colleges teamed with NC State startup company Locus Biosciences to test the effectiveness of using a virus called a bacteriophage to carry a programmable CRISPR to specifically target and eliminate C. difficile bacteria, a search-and-destroy mission that holds promise for human gut health.

“We wanted to engineer phages with self-targeting CRISPR payloads and deliver them to the gut of an organism of choice – in this case a mouse – in order to have a beneficial impact on host health and to prevent disease,” said Rodolphe Barrangou, the Todd R. Klaenhammer Distinguished Professor of Food, Bioprocessing and Nutrition Sciences at NC State and co-corresponding author of a paper describing the research.

Co-corresponding author Casey M. Theriot, an assistant professor of infectious disease at NC State, said that use – and overuse – of antibiotics increases susceptibility to C. difficile infection, as antibiotics wipe out both good and bad bacteria in the gut. Relapses occur in some 30% of human patients treated with a standard antibiotic to eliminate C. difficile.

“We need to target the precise pathogen without disturbing the rest of the microbiome, and that’s what this approach does,” she said.

CRISPR technologies have been used to precisely remove or cut and replace specific genetic code sequences in bacteria. The CRISPR method used in this study involved Cas3 proteins that acted like an arcade game Pac-Man, Barrangou said, chomping C. difficile bacteria and causing extensive DNA damage.

In the lab, the CRISPR-Cas systems effectively killed C. difficile bacteria. After that, the researchers tested this approach in mice infected with C. difficile. Two days after the CRISPR treatment, the mice showed reduced C. difficile levels, but those levels grew back two days later.

“C. difficile is really difficult to work with, hence its name,” Theriot said.

“This was a positive first step in a long process,” Barrangou said. “The results of using phages to deliver CRISPR payloads open up new avenues for other infectious diseases and beyond.”

Next steps include retooling the phage to prevent C. difficile from returning after the initial effective killing. The researchers said that future work will also involve developing a library of different phages for various C. difficile strains.

The work was funded by Locus Biosciences. Barrangou was chief scientific officer of the company after co-founding it. Theriot is a scientific advisor to the company. Lead co-author Kurt Selle worked in Barrangou’s lab as a graduate student; lead co-author Joshua Fletcher works in the Theriot lab as a postdoctoral fellow. Other co-authors include Hannah Tuson, Daniel Schmidt, Lana McMillan, Gowrinarayani S. Vridhambal, and David G. Ousterout from Locus Biosciences; Alissa Rivera from NC State; Stephanie Montgomery from UNC-Chapel Hill; and Louis-Charles Fortier from Universite de Sherbrooke in Canada.

Note to editors: An abstract of the paper follows.

“In vivo targeting of Clostridioides difficile using phage-delivered CRISPR-Cas3 antimicrobials”

Authors: Joshua R. Fletcher, Alissa J. Rivera, Rodolphe Barrangou, Casey M. Theriot, NC State University; Kurt Selle, Hannah Tuson, Daniel S. Schmitt, Lana McMillan, Gowrinarayani S. Vridhambal and David G. Ousterout, Locus Biosciences; Stephanie A. Montgomery, University of North Carolina-Chapel Hill; and Louis-Charles Fortier, Universite de Sherbrooke

Published: Online March 10, 2020 in mBio

DOI: 10.1128/mBio.00019-20

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 U.S. 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 treatment of CDI, likely contributing to high recurrence rates due to 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 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 obligately lytic is feasible and contributes to therapeutic suitability of intrinsic C. difficile phages, despite specific challenges encountered in the disease phenotypes of phage treated animals. Our findings suggest that phage-delivered programmable CRISPR therapeutics have the potential to leverage the specificity and apparent safety of phage therapies, and improve their potency and reliability for eradicating specific bacterial species within complex communities, offering a novel mechanism to treat pathogenic and/or multidrug-resistant organisms.