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PLoS One

Title:		Engineering conjugative CRISPR-Cas9 systems for the targeted control of enteric pathogens and antibiotic resistance
Author(s):		Sheng H; Wu S; Xue Y; Zhao W; Caplan AB; Hovde CJ; Minnich SA;
Address:		"Animal, Veterinary and Food Science, University of Idaho, Moscow, Idaho, United States of America. Department of Chemical and Biological Engineering, University of Idaho, Moscow, Idaho, United States of America. Department of Plant Sciences, University of Idaho, Moscow, Idaho, United States of America"
Journal Title:		PLoS One
Year:		2023
Volume:		20230912
Issue:		9
Page Number:		e0291520 -
DOI:		10.1371/journal.pone.0291520
ISSN/ISBN:		1932-6203 (Electronic) 1932-6203 (Linking)
Abstract:		"Pathogenic Escherichia coli and Salmonella enterica pose serious public health threats due to their ability to cause severe gastroenteritis and life-threatening sequela, particularly in young children. Moreover, the emergence and dissemination of antibiotic resistance in these bacteria have complicated control of infections. Alternative strategies that effectively target these enteric pathogens and negate or reduce the need of antibiotics are urgently needed. Such an alternative is the CRISPR-Cas9 system because it can generate sequence-specific lethal double stranded DNA breaks. In this study, two self-transmissible broad host range conjugative plasmids, pRK24 and pBP136, were engineered to deliver multiplexed CRSIPR-Cas9 systems that specifically target Enterohemorrhagic and Enteropathogenic strains of E. coli (EHEC and EPEC), S. enterica, and blaCMY-2 antibiotic resistance plasmids. Using in vitro mating assays, we show that the conjugative delivery of pRK24-CRISPR-Cas9 carrying guide RNAs to the EPEC/EHEC eae (intimin) gene can selectively kill enterohemorrhagic E. coli O157 eae+ cells (3 log kill at 6 h) but does not kill the isogenic Deltaeae mutant (P<0.001). Similar results were also obtained with a pBP136 derivative, pTF16, carrying multiplexed guide RNAs targeting E. coli eae and the S. enterica ssaN gene coding for the type III secretion ATPase. Another pBP136 derivative, TF18, carries guide RNAs targeting S. enterica ssaN and the antibiotic resistance gene, blaCMY-2, carried on the multi-drug resistant pAR06302. Introduction of pTF18 into bacteria harboring pAR06302 showed plasmids were cured at an efficiency of 53% (P<0.05). Using a murine neonate EPEC infection model, pTF16 was delivered by a murine derived E. coli strain to EPEC infected mice and showed significant reductions of intestinal EPEC (P<0.05). These results suggest that establishing conjugative CRISPR-Cas9 antimicrobials in the intestinal microbiome may provide protection from enteric pathogens and reduce antibiotic resistance without disrupting the normal microbiota"
Keywords:		"Animals Mice CRISPR-Cas Systems/genetics Engineering *Gastroenteritis *Enterohemorrhagic Escherichia coli/genetics Anti-Bacterial Agents/pharmacology Drug Resistance, Microbial *Escherichia coli O157;"
Notes:		"MedlineSheng, Haiqing Wu, Sarah Xue, Yansong Zhao, Wei Caplan, Allan B Hovde, Carolyn J Minnich, Scott A eng P20 GM103408/GM/NIGMS NIH HHS/ Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. 2023/09/12 PLoS One. 2023 Sep 12; 18(9):e0291520. doi: 10.1371/journal.pone.0291520. eCollection 2023"