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The intestinal epithelium is continuously renewed by intestinal epithelial stem cells (IESCs) positioned at the base of each crypt. Mesenchymal-derived factors are essential to maintain IESCs; however, the cellular composition and development of such mesenchymal niche remains unclear. Here, we identify pericryptal CD34⁺ Gp38⁺ αSMA⁻ mesenchymal cells closely associated with Lgr5⁺ IESCs. We demonstrate that CD34⁺ Gp38⁺ cells are the major intestinal producers of the niche factors Wnt2b, Gremlin1, and R-spondin1, and are sufficient to promote maintenance of Lgr5⁺ IESCs in intestinal organoids, an effect mainly mediated by Gremlin1. CD34⁺ Gp38⁺ cells develop after birth in the intestinal submucosa and expand around the crypts during the third week of life in mice, independently of the microbiota. We further show that pericryptal CD34⁺gp38⁺ cells are rapidly activated by intestinal injury, up-regulating niche factors Gremlin1 and R-spondin1 as well as chemokines, proinflammatory cytokines, and growth factors with key roles in gut immunity and tissue repair, including IL-7, Ccl2, Ptgs2, and Amphiregulin. Our results indicate that CD34⁺ Gp38⁺ mesenchymal cells are programmed to develop in the intestine after birth to constitute a specialized microenvironment that maintains IESCs at homeostasis and contribute to intestinal inflammation and repair after injury.

Escherichia coli is a ubiquitous gut commensal but also an opportunistic pathogen responsible for severe intestinal and extra-intestinal infections. Shiga toxin-producing E. coli (STEC) pose a significant public health threat, particularly in children, where infections can lead to bloody diarrhea and progress to hemolytic uremic syndrome (HUS), a life-threatening condition with long-term complications. Antibiotics are contraindicated in STEC infections due to their potential to induce prophages carrying Shiga toxin (stx) genes, triggering toxin production. Here, we present a CRISPR-based antimicrobial strategy that selectively targets and eliminates O157 STEC clinical isolates while preventing toxin release. We designed a Cas12 nuclease to cleave >99% of all stx variants found in O157 strains, leading to bacterial killing and suppression of toxin production. To enable targeted delivery, we engineered a bacteriophage-derived capsid to specifically transfer a non-replicative DNA payload to E. coli O157, preventing its dissemination. In a mouse STEC colonization model, our therapeutic candidate, EB003, reduced bacterial burden by a factor of 3x103. In an infant rabbit disease model, EB003 mitigated clinical symptoms, abrogated stx-mediated toxicity, and accelerated epithelial repair at therapeutically relevant doses. These findings demonstrate the potential of CRISPR-based antimicrobials for treating STEC infections and support further clinical development of EB003 as a precision therapeutic against antibiotic-refractory bacterial pathogens.Competing Interest StatementAll authors are current or former employees, or paid advisors, of Eligo Bioscience. Eligo Bioscience owns US patent nos. US11,905,516, US10,808,254, US11,078,490, US11,946,056, US11,661,443, US11,236,133, US11,512,116, US11584918, US11970716, US11746352, US12,098,372, and US11584781; Japanese patent No. JP7250702; Japanese patent No. JP7627223; Korean patent No. KR10-2563835; Israel patent No. 267932; and international patent application Nos. WO2018/141907, WO2020/109339, WO2020/187836, WO2022/144381 and WO2022/144382 relating to certain research described in this article.Footnotes* https://enterobase.warwick.ac.uk/species/index/ecoli* https://www.ncbi.nlm.nih.gov/bioproject/PRJNA248042/* https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA259645