A team of researchers at the Korea Research Institute of Bioscience and Biotechnology (KRIBB) has developed a CRISPRi-based plasmid selection system by building a cascaded logic inverter to mimic antibiotics and thereby reduce antibiotic-resistance genes.

The first author Dr. Kim Seong-keun (left), and research director Lee Dae-hee of Korea Research Institute of Bioscience and Biotechnology (KRIBB)'s Synthetic Biology Research Center demonstrate the building of the synthetic biological circuit in the lab. (Credits: KRIBB)
The first author Dr. Kim Seong-keun (left), and research director Lee Dae-hee of Korea Research Institute of Bioscience and Biotechnology (KRIBB)'s Synthetic Biology Research Center demonstrate the building of the synthetic biological circuit in the lab. (Credits: KRIBB)

Antibiotic screening is most widely used to select plasmids that will survive stably in host cells when developing a mass production manufacturing process of bio-products. However, continued use of antibiotics increases antibiotic resistance, induces allergic reactions, and raises manufacturing costs.

As an alternative, nutritional-required strains can be used. However, it is difficult to make strains, and this method has a low screening ability. Plus, there is a limit to the number of genes available for screening.

For example, in biopharmaceuticals such as DNA vaccines, the smaller the size of the plasmid, the higher the transfer efficiency in the human body, and the higher the amount of antigen expression. Thus, a platform capable of selecting small plasmids is required.

The diagram shows the design of the CRISPRi-based inverter cascade where (A) the action of antibiotic-resistance genes against antibiotics can be interpreted as an inverter (NOT gate) cascade in synthetic biology and (B) CRISPRi-based inverter cascades can mimic the relationship between antibiotics and antibiotic-resistance genes. (Credit: Nucleic Acids Research journal)
The diagram shows the design of the CRISPRi-based inverter cascade where (A) the action of antibiotic-resistance genes against antibiotics can be interpreted as an inverter (NOT gate) cascade in synthetic biology and (B) CRISPRi-based inverter cascades can mimic the relationship between antibiotics and antibiotic-resistance genes. (Credit: Nucleic Acids Research journal)

Accordingly, the research team developed a system that only selects cells with plasmids by creating an intelligent gene circuit based on synthetic biology. The principle is that cells survive and are selected because interference by gene scissors does not occur in the presence of plasmids. They combined a NOT logic circuit and CRISPR interference (CRISPRi) gene that suppresses the expression of a specific gene to replace the function of the antibiotic.

The developed system can be expanded to simultaneously select multiple plasmids and can be used to develop DNA vaccines by using very small guide RNAs with 160 base pairs rather than antibiotic resistance genes as a plasmid screening marker.

"Antibiotics have been used for a long time to maintain plasmids and screen for recombinant microorganisms but at the risk of creating resistant bacteria," Professor Lee Dae-hee who led the study said. "If synthetic biology is applied to the antibiotic-free bio-manufacturing process in the future, it can be used in various industries including the bioindustry."

The study was published in the online edition of Nuclear Acids Research in Synthetic Biology and Bioengineering on December 13, 2022.

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