Comparative Evaluation of Engineered Bacteria and Yeast for Oral Delivery of CRISPR/Cas9 Systems in Colon Cancer Therapy

X33, which upon colonization in the intestine, secreted extracellular vesicles carrying the Cas9 protein and ART1-targeting sgRNA for tumor-specific gene disruption. Arginine ADP-ribosyltransferase 1 (ART1) plays a crucial role in the biological regulation of colon cancer, which was for the first time to the best of our knowledge, employed in vivo as a target gene in this study. Furthermore, we employed polydopamine (PDA) coating and gastrointestinal synthetic epithelial lining systems to facilitate microbial viability and intestinal retention, establishing on site cell factories for sustained CRISPR secretion. In subcutaneous tumor-bearing murine models, both delivery systems demonstrated comparable antitumor efficacy with significant tumor suppression. Taken together, the genetically modified microbial platform using bacterial and yeast strategies shows great potential and broad therapeutic versatility, offering a promising CRISPR-based solution for CRC treatment.