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17 December 2018 by Julie Monroe

Genetic Circuits: Reprogramming Biology

Science has long been a field driven by curiosity. In more recent history, pressure has escalated for science to offer not just new discoveries and ideas, but ways to implement them for the betterment of society, a role in which engineers excel.

Scientists and engineers are two groups that are often teased about their unique ways of looking at the same problem. Jokes aside, their differing perspectives have contributed to the evolution of novel technologies that hold the potential to revolutionize medicine, agriculture, and numerous other disciplines.

One of these novel technologies is the creation of synthetic genetic circuits.

Genetic circuits can be compared to circuits used in computer science applications. A sensor receives input that is processed through components called gates, which then generate outputs or perform functions.

Cells contain similar components as a computer circuit, albeit in chemical form, that produce biological function. The goal of genetic circuits in synthetic biology is opposite of the top-down genomics approach to genetic engineering. Instead, it is to forward engineer systems from the ground up that can be inserted into cells for novel functions.

The first genetic circuits, inserted into E. coli, were very simple and consisted of toggle switches and oscillators. Toggle switches are formed through the presence of two mutual inhibitory proteins.

For example, Gene A wants to deactivate Gene B, and vice versa. The key is to isolate a chemical or environmental trigger that will cause the active Gene A to stop expression, so that inactive Gene B will be free to begin expression. After the trigger is gone, Gene B will remain active and suppress Gene A.

Oscillators are described as blinking circuits and work in a ring fashion. Gene A is triggered to begin suppressing Gene B; Gene B works to suppress Gene C, and Gene C works to suppress Gene A. Although elementary, these two basic circuits have the potential to serve as sensors for environmental chemicals or pathogens.

In a therapeutic scenario, for instance, genetic switches that turn on gene expression could be engineered in bacteriophages to break down antibiotic resistant biofilms.

The creation of complex, sophisticated cellular level circuits has proven to be a huge undertaking. Not all proteins are understood well enough to accurately predict expression, especially under changing cell conditions. Unlike electronic circuits, where components are physically separate, the chemical components of a genetic circuit are constantly interacting and possibly interfering with each other. Finally, it is difficult ascertain if an activated genetic circuit is operating correctly over time.

Researchers at MIT have been able to move from simple circuits to multiplex ones through the development of a software that can design layered genetic circuits and predict their experimental outcomes.

To do this, they utilized an older programming language that was originally developed for electronic chips. “We went in and hacked [Verilog] so that instead of compiling a circuit to silicon, it would compile it to DNA,” says Dr. Christopher Voigt, Professor of Chemical Engineering at MIT.

The software takes a preliminary circuit concept imagined by a researcher and designs a genetic circuit schematic that accounts for possible protein interference. It spits out a circuit-coded DNA sequence that can be sent off to a DNA synthesis company to be compiled.

Along with advances like the Verilog software (now called Cello), technologies like CRISPR are making synthetic biology processes much easier and more efficient. In the original CRISPR/Cas9 system, DNA could be cleaved at a particular locus with the use of a Cas9/gRNA (guide RNA) complex.

For genetic circuits, researchers discovered that gene expression can be modulated by sending a deactivated Cas9 (dCas9) to complex with a gRNA. The dCas9 no longer has endonuclease activity to cleave the DNA, but is instead used as a DNA targeting molecule. When fused to transactivation and repression protein domains it has the ability to influence activation and repression of target gene transcription.

This modified system is referred to as CRISPRi (interference) or CRISPRa (activation). Because of CRISPR’s talents, researchers believe that it will benefit synthetic biology circuit design in several ways. Here are just a few examples:

  • Engineering of microbial pathogens with repressed virulent or antibiotic resistant genes.
  • Generation of therapeutic circuits for diseases that have multiple gene targets.
  • Expediting genomics research and understanding of how genes interact to result in phenotypes.

Timothy Lu, Associate Professor of Biological Engineering, Electrical Engineering, and Computer Science at MIT, believes that these great advances over the last decade have developed because of a convergence of interest from various fields to the possibilities of synthetic biology.

“What’s really allowed this field to take off is the influx of people from pretty nontraditional backgrounds: people like physicists, engineers, non-biological scientists…who are interested in understanding biology and manipulating it,” said Lu in a 2014 interview. “On the other hand, there’s been a technological advancement in our ability to read and write DNA, and that’s given us the raw tools that we need for understanding a biological system and then reprogramming it for new functions.”