关键词: 生物安全防控, 营养缺陷, 自杀基因, 毒素-抗毒素, 基因编辑, 非天然核苷酸, 非天然氨基酸

Abstract:

With the rapid rise of synthetic genes and engineered bacteria in disease diagnosis and treatment, it poses a growing risk of biosafety issues. We review the biocontainment strategies based on synthetic biology, and especially highlight the recent biocontainment studies on diagnosis or therapeutic bacteria. There are several goals of biocontainment. One is to reduce the escapee rate of engineered bacteria by limiting the survival within biological barriers. The second is to prevent synthetic genes transferring from engineered bacteria into environment. Auxotrophy and kill-switches are widely applied in biocontainment of engineered bacteria. Auxotrophic organisms with essential genes knockout rely on key metabolites supplement for survival. Kill-switches are inducible toxic gene circuits, such as suicide switch and toxin-antitoxin system. Once engineered bacteria leave from human bodies, the toxic genes are switched on to kill organisms. Genetic separation and DNA breaking are useful strategies to keep synthetic genes from spreading into environmental organisms. Essential genes and genes of interest can be distributed into multiple vectors or chromosomes, each vector or chromosome depends on the others for replication. DNA breaking technologies like CRISPR or other DNA nucleases are used to digest chromosome or plasmid inside engineered bacteria, which regulates host survival and synthetic gene transferring. The cross-feeding of environmental metabolites and genes into engineered bacteria could lead to biocontainment failure. Several unnatural nucleic acids have been developed for gene replication and transcription, and much more unnatural amino acids are deployed for protein translation. One advantage of these unnatural systems is the orthogonality, which prevent synthetic gene transfer to natural organism. The chemically synthesized unnatural nucleic acid and amino acids are not present in environment, so the synthetic auxotrophy overcomes the cross-feeding limitation of natural auxotrophy. Biocontainment systems with multiple-layered design based on different synthetic biological principles have the potentials to solve the biosafety issues in the future.

Key words: biocontainment, auxotrophy, suicide gene, toxin-antitoxin, gene editing, unnatural nucleic acid, unnatural amino acid