TY - JOUR T1 - Cas9-Assisted Biological Containment of a Genetically Engineered Human Commensal Bacterium and Genetic Elements JF - bioRxiv DO - 10.1101/2021.11.03.467106 SP - 2021.11.03.467106 AU - Naoki Hayashi AU - Yong Lai AU - Mark Mimee AU - Timothy K. Lu Y1 - 2021/01/01 UR - http://biorxiv.org/content/early/2021/11/03/2021.11.03.467106.abstract N2 - Sophisticated gene circuits built by synthetic biology can enable bacteria to sense their environment and respond predictably. Biosensing bacteria can potentially probe the human gut microbiome to prevent, diagnose, or treat disease. To provide robust biocontainment for engineered bacteria, we devised a Cas9-assisted auxotrophic biocontainment system combining thymidine auxotrophy, an Engineered Riboregulator (ER) for controlled gene expression, and a CRISPR Device (CD). The CD prevents the engineered bacteria from acquiring thyA via horizontal gene transfer, which would disrupt the biocontainment system, and inhibits the spread of genetic elements by killing bacteria harboring the gene cassette. This system tunably controlled gene expression in the human gut commensal bacterium Bacteroides thetaiotaomicron, prevented escape from thymidine auxotrophy, and blocked transgene dissemination for at least 10 days. These capabilities were validated in vitro and in vivo. This biocontainment system exemplifies a powerful strategy for bringing genetically engineered microorganisms safely into biomedicine.Competing Interest StatementT.K.L. is a co-founder of Senti Biosciences, Synlogic, Engine Biosciences, Tango Therapeutics, Corvium, BiomX, Eligo Biosciences, Bota.Bio, and Avendesora. T.K.L. also holds financial interests in nest.bio, Ampliphi, IndieBio, MedicusTek, Quark Biosciences, Personal Genomics, Thryve, Lexent Bio, MitoLab, Vulcan, Serotiny, and Avendesora. N.H is an employee of JSR Corporation. Other authors declare no competing interests. ER -