biostudies-literatureDion MBNatural Sciences and Engineering Research Council of CanadaNovo Nordisk FoundationCanada First Research Excellence FundTier 1 Canada Research Chair in BacteriophagesIntestinal Microbiomics, Institute of Nutrition, Metabolism, and DiabetesFaculty of Health and Medical Sciences, University of CopenhagenDanish Agency for Science and Higher EducationCanadian Institutes of Health ResearchCIHRCalcul QuébecFonds de Recherche du Québec-Nature et TechnologiesBRIDGE–Translational Excellence ProgrammeJoint Programming Initiative “Healthy Diet for a Healthy Life,”Digital Research Alliance of CanadaNovo Nordisk Fondenwrae005https://www.ebi.ac.uk/biostudies/studies/S-EPMC10910852biostudies-literatureUnknown18(1)CRISPR-Cas systems are defense mechanisms against phages and other nucleic acids that invade bacteria and archaea. In Escherichia coli, it is generally accepted that CRISPR-Cas systems are inactive in laboratory conditions due to a transcriptional repressor. In natural isolates, it has been shown that CRISPR arrays remain stable over the years and that most spacer targets (protospacers) remain unknown. Here, we re-examine CRISPR arrays in natural E. coli isolates and investigate viral and bacterial genomes for spacer targets using a bioinformatics approach coupled to a unique biological dataset. We first sequenced the CRISPR1 array of 1769 E. coli isolates from the fecal samples of 639 children obtained during their first year of life. We built a network with edges between isolates that reflect the number of shared spacers. The isolates grouped into 34 modules. A search for matching spacers in bacterial genomes showed that E. coli spacers almost exclusively target prophages. While we found instances of self-targeting spacers, those involving a prophage and a spacer within the same bacterial genome were rare. The extensive search for matching spacers also expanded the library of known E. coli protospacers to 60%. Altogether, these results favor the concept that E. coli's CRISPR-Cas is an antiprophage system and highlight the importance of reconsidering the criteria use to deem CRISPR-Cas systems active.The ISME journalEscherichia coli CRISPR arrays from early life fecal samples preferentially target prophages.PMC109108522019-05183950-232136NNF20OC0061029143924259257NNF18SA0034956Shah SAThorsen JDion MBKrogfelt KAAllard APetit MAStokholm JDeng LSchjorring SHorvath PNielsen DSMoineau SfalseEscherichia coli CRISPR arrays from early life fecal samples preferentially target prophages.CRISPR-Cas systems are defense mechanisms against phages and other nucleic acids that invade bacteria and archaea. In Escherichia coli, it is generally accepted that CRISPR-Cas systems are inactive in laboratory conditions due to a transcriptional repressor. In natural isolates, it has been shown that CRISPR arrays remain stable over the years and that most spacer targets (protospacers) remain unknown. Here, we re-examine CRISPR arrays in natural E. coli isolates and investigate viral and bacterial genomes for spacer targets using a bioinformatics approach coupled to a unique biological dataset. We first sequenced the CRISPR1 array of 1769 E. coli isolates from the fecal samples of 639 children obtained during their first year of life. We built a network with edges between isolates that reflect the number of shared spacers. The isolates grouped into 34 modules. A search for matching spacers in bacterial genomes showed that E. coli spacers almost exclusively target prophages. While we found instances of self-targeting spacers, those involving a prophage and a spacer within the same bacterial genome were rare. The extensive search for matching spacers also expanded the library of known E. coli protospacers to 60%. Altogether, these results favor the concept that E. coli's CRISPR-Cas is an antiprophage system and highlight the importance of reconsidering the criteria use to deem CRISPR-Cas systems active.2024-01-01T00:00:00Z2024 Jan2026-06-28T03:08:20.006Z2025-04-05T11:38:33.779ZS-EPMC109108523836619210.1093/ismejo/wrae005