One step closer to efficient gene regulation in vitro: T7 RNAP

Genetic engineering focuses not only on gene editing – a lot of work is dedicated to gene regulation. Specific genes can be activated or deactivated, with reversibility and temporary nature in mind. As an example, US’ DARPA is working on genetic drugs, which will boost radiation resistance by changes in gene regulation (covered here).

Scientists from Harvard University developed a new kind of tool, aimed at changing gene regulation outside of cells – in vitro.

Their approach was inspired by natural molecules called transcription factors. Those proteins govern fundamentals of gene regulation. They have the ability to bind specific places in the genome. After binding, they affect nearby genes by boosting or decreasing the expression. Cells live thanks to long cascades of transcription factors activating and deactivating many genes in response to changes in the environment and in the inner metabolism.

Artificial counterparts of transcription factors are not new. Scientists work on them for decades, and recently they began to create a variety of new ones. Described approach fits into this field, but with one important twist – new artificial transcription factors can be programmed by a single-stranded DNA, which makes them relatively easy and cheap to use.

Researchers used RNA polymerase from T7 bacteriophage. Its state was defined as “caged” (gene deactivation) and “uncaged” (gene activation). The regulation – regulatory input – relied on nucleic acids.

In the experimental application of the system, scientists developed regulatory networks in vitro – outside of a living cell. These included feedback circuits, cascades, as well as transcriptional multiplexing.

There are important limitations to the technology. For instance the “uncaging” is irreversible and the circuits are prone to RNases/DNases present in cell-free extracts. Nevertheless, the study presents an important step forward for synthetic biology and its many applications.

Publication: Chou, L. Y., & Shih, W. M. (2019). In vitro transcriptional regulation via nucleic-acid-based transcription factors. ACS Synthetic Biology.
Photo: Eva Nogales, Berkeley Lab.

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