Crispr-edited commensals of the nasopharynx as platform for developing next-generation microbial probiotics

Abstract

The nasopharynx is a critical mucosal barrier where commensals sustain immune balance and block pathogen colonization. Corynebacterium accolens and Dolosigranulum pigrum are linked to reduced pneumococcal carriage, fewer viral–bacterial coinfections, and modulation of host immunity. Their metabolites—short-chain fatty acids, nitric oxide, bacteriocins—reinforce epithelial integrity and regulate NF-κB, JAK/STAT, TGF-β/STAT3, promoting Treg cells. Natural effects remain insufficient for therapy. CRISPR/Cas now enables programming these commensals into “living probiotics” that deliver therapeutic molecules in a context-specific manner. Objective: To evaluate C. accolens and D. pigrum as microbial therapy candidates and propose a CRISPR/Cas9 framework for engineering probiotics to modulate mucosal immunity, exclude pathogens, and transmit anti-inflammatory signals. Methods: A systematic review (2005–2025) was conducted in PubMed, Scopus, Web of Science, EMBASE, Google Scholar using terms: “nasopharyngeal microbiome,” “C. accolens,” “D. pigrum,” “CRISPR editing,” “probiotics,” “mucosal immunity.” Eligible studies covered genomic, proteomic, metabolomic features, immune interactions, CRISPR editing in probiotics, and preclinical/in silico models. Excluded: unrelated niches, insufficient methods, and non-CRISPR work. Data were synthesized into five topics: ecological roles, molecular/secretome features, CRISPR advances, biosafety design, and validation strategies. Results: Both taxa emerged as keystone members of the nasopharyngeal microbiome. Genomics revealed stable chromosome organization, no virulence genes, and pathways for SCFA biosynthesis, lipid metabolism, and adhesion. Proteomic and metabolomic studies confirmed secretion of antimicrobial peptides, bacteriocins, and nitric oxide, reducing pathogens and modulating immunity. Functional assays showed TLR2/6 activation, NF-κB inhibition, STAT3/TGF-β promotion, and IL-10 induction, strengthening barriers, enhancing IgA, and favoring regulatory immunity. A CRISPR design was proposed for C. accolens with an IL-10 cassette under an inflammation-inducible promoter plus kill-switches, auxotrophy, and self-limiting nuclease activity. In silico modeling confirmed ecological compatibility and circuit stability. Validation was outlined across in silico prediction, in vitro epithelium–MALT co-cultures, and in vivo murine models with checkpoints for genomic stability, tolerance, and absence of systemic spread. Personalization included microbiome-guided strain selection, prebiotic co-formulation, and biofilm assays, ensuring therapeutic expression only under inflammation. Conclusions: CRISPR-edited nasopharyngeal commensals are a promising therapeutic platform. C. accolens and D. pigrum combine stability, safety, and intrinsic immunomodulatory capacity, amplified by precise engineering. Inflammation-inducible IL-10 circuits enable localized, context-specific mucosal immunomodulation. Biosafety modules minimize ecological risk and secure evolutionary stability. Such engineered strains may deliver robust, personalized, clinically relevant interventions for infectious and inflammatory airway diseases.