KAIST

<(From left to right) Dr. Soo Young Moon, KAIST Institute of Life Science,Professor Ju Young Lee, Graduate School of Engineering Biology (Adjunct Professor of Biological Sciences),Dr. Myung Hyun Noh, Korea Research Institute of Chemical Technology (KRICT),Researcher Nan-Yeong An, Department of Biological Sciences>

Turning genes on and off is like flipping a light switch, controlling whether genes in a cell are active. When a gene is turned on, the production of proteins or other substances is promoted; when it's turned off, production is suppressed. Korean researchers have gone beyond the limitations of existing CRISPR technology, which focused primarily on "off" functions, by developing the world's first innovative system that can simultaneously turn genes on and off, opening a new paradigm for the synthetic biology-based bio-industry.

A joint research team led by Professor Ju Young Lee of KAIST Graduate School of Biological Engineering (Adjunct Professor of Biological Sciences) and Dr. Myung Hyun Noh of the Korea Research Institute of Chemical Technology (KRICT), an organization under the National Research Council of Science & Technology (NST) , announced on the 21st that they have developed a new dual-mode CRISPR gene editing system that can simultaneously turn on and off desired genes in E. coli.

E. coli is a representative microorganism that is easy to experiment with and can be directly applied to industrial uses. Meanwhile, CRISPR technology is considered one of the most innovative tools in 21st-century biotechnology.

In particular, bacteria, which are the foundation of synthetic biology, have a simple structure and multiply rapidly, while also being able to produce a variety of useful substances. Therefore, gene activation in bacteria is a key technology for designing "microbial factories," and its industrial value is very high.

The core of synthetic biology is to design the genetic circuits of living organisms like programming a circuit board to perform a desired function. Just as switches are turned on and off in an electronic circuit, a technology is needed to optimize metabolic pathways by activating certain genes while suppressing others. The dual-mode gene scissors developed by the research team are the key tool that enables this precise gene regulation.

Existing CRISPR gene scissors were primarily specialized for the "off" function (repression) and were excellent at blocking gene expression, but their ability to turn genes on was very limited.

Furthermore, for CRISPR to work, a specific DNA recognition sequence (PAM, protospacer adjacent motif) is required, and the narrow range of PAM recognition in existing systems limited the scope of genes that could be controlled.

In addition, while CRISPR-based activation (CRISPRa) has been somewhat developed in eukaryotic cells (human, plant, and animal cells), there were limitations in bacteria where the "on" function did not work properly due to differences in their internal transcription regulation mechanisms.

To overcome these limitations, the research team expanded the target range to access more genes and significantly improved gene activation performance by utilizing E. coli proteins. As a result, the gene scissors, which were previously "mainly for turning off," have evolved into a system that can simultaneously control both "on" and "off."

The performance verification results of the developed system were very impressive. In gene activation experiments, expression levels increased by up to 4.9 times, and in repression experiments, they could be suppressed by up to 83%.

Even more astonishing was the ability to control two different genes simultaneously. The team successfully activated one gene by 8.6 times while simultaneously repressing another by 90%.

< (Left) The principle of the dual-mode CRISPR gene scissors. When the guide RNA (gRNA) binds to the target sequence, dxCas9-CRP either promotes (CRISPRa) or inhibits (CRISPRi) the binding of RNA polymerase near the transcription start site, precisely controlling gene expression. (Center) A large-scale screening of the entire E. coli genome is conducted to identify key regulatory targets for optimizing target substance production. The metabolic pathway for producing the target substance is then re-engineered by simultaneously regulating gene expression through activation and repression. (Right) The dual-mode CRISPR gene scissors system enables systematic redesign of cell metabolism, precise reconfiguration of gene expression, and the construction of microbial strains that can perform various functions, ultimately leading to a significant increase in target substance productivity. In this study, the dual-mode CRISPR system was applied to E. coli to demonstrate the enhanced production of 'violacein,' a purple functional biopigment with anticancer effects, and its potential for expansion to other bacterial species was also confirmed. >

To demonstrate the practicality of this technology, the research team challenged themselves to increase the production of 'violacein,' a purple pigment with anticancer properties. Through large-scale experiments on all genes of E. coli, they identified genes that help in violacein production.

As a result, production increased by 2.9 times when the 'rluC' gene, which helps protein production, was turned on, and by 3.0 times when the 'ftsA' gene, which helps cell division, was turned off. When both genes were controlled simultaneously, a greater synergistic effect was observed, achieving a remarkable 3.7-fold increase in production.

Dr. Myung Hyun Noh of KRICT stated, "Precise gene activation is now possible in bacteria," and "This will greatly contribute to the development of the synthetic biology-based bio-industry."

Professor Ju Young Lee said, "This research is a successful outcome of combining gene scissors with synthetic biology to significantly enhance the efficiency of microbial production platforms," and "The ability to control a complex genetic network with a single system presents a new research paradigm." He added, "This technology has also been confirmed to work in other bacterial species and can be utilized in various fields such as the production of biopharmaceuticals, chemicals, and fuels."

< (A) A diagram of the violacein biosynthesis pathway, a functional biopigment produced from the starting material L-tryptophan through several enzymatic reactions. Violacein is a functional substance with broad applications in various industries and research fields, including medicine, healthcare, dyes, textiles, food and beverage, and cosmetics. (B) The results of a large-scale screening of gRNAs for gene activation and repression using the dual-mode CRISPR gene scissors system confirmed a 2.9-fold increase in violacein production (mg/L) upon rluC activation and a 3.0-fold increase upon ftsA repression compared to the control group. >

The results of this research, with Dr. Soo Young Moon, a postdoctoral researcher at our university's Institute of Life Science, as the first author, were published online in 'Nucleic Acids Research,' a top-tier journal in the field of molecular biology, on August 21st.

• Paper Title: Dual-mode CRISPRa/i for genome-scale metabolic rewiring in Escherichia coli

• Author Information: Soo Young Moon (KAIST, First Author), Mi Ri Kim (KRICT), Nan-Yeong An (KAIST), Myung Hyun Noh (KRICT, Corresponding Author), Ju Young Lee (KAIST, Corresponding Author) (Total of 5 authors)

• DOI: 10.1093/nar/gkad818

This research was supported by the joint research and development program of the Ministry of Science and ICT, the National Research Foundation of Korea, and Boston Korea.