Taylor Sheahan - June 20, 2018

Chris Isaac at the Meeting of States Parties to the Biological Weapons Convention (BWC) in Geneva, Switzerland.

In 2017, the University of Lethbridge Collegiate iGEM team aimed to lower barriers to synthetic biology to the public, by developing a modular and safe cell-free platform. Cell-free approaches to biological engineering provide a promising alternative to cell-based systems. This is due to their reduced complexity and potential for bio-contamination, as wells as novel engineering capabilities, such as the ability to incorporate non-canonical amino acids and toleration of toxic compounds. Due to the inherent safety of cell-free platforms, the team envisioned such a system would provide a powerful tool as an educational platform, which could be used to teach students the basics of synthetic biology. Additionally, the cell-free platform would be accessible to the public, enabling "DIY" enthusiasts and biotechnology start-ups. 

Although the potential for cell-free platforms is high, it is necessary to address the potential misuse of cell-free systems and develop a strategy to mitigate the threat of suspicious users. This is where Chris Isaac, a member of the U of L iGEM team, comes in. Isaac identified that the flexibility of cell-free systems makes them highly amenable to genetic recoding, where canonical relationships between codons and anticodons are not necessarily consistent. Thus, there is the potential for dangerous users to “encrypt” sequences that, when synthesized using a novel genetic code, will produce a harmful compound. To address this threat, Isaac aimed to develop software that would identify recoded sequences to ensure that they will not be synthesized. At the 2017 iGEM Giant Jamboree, Isaac and the team were acknowledged for their ability to identify biosecurity risks associated with cell-free systems, as well as for their initiative taken to develop mitigation strategies. 

Building on the momentum from the Giant iGEM Jamboree, Isaac was selected along with four other students to travel to the UN campus as an iGEM delegate. In the winter of 2017, the delegates attended the intersessional Meeting of the States Parties to the Biological Weapons Convention (BWC) in Geneva, Switzerland. 

“At the convention, we were able to attend the plenary sessions and listen to statements made by the parties” says Isaac. “While these statements appeared to be largely formality, they were in fact glimpses into long-standing issues, brief remarks on current events, and calls to action for the continued support of the BWC.” 

The delegates had the opportunity to discuss biosecurity and synthetic biology with representatives from around the globe, creating an open dialogue between young scientists and regulatory officials. 

“The experience provided a great look into how governments prevent and control biological threats, and was also a good introduction to the non-governmental agencies who are working on these problems.”

Continuing his work towards addressing biosecurity threats associated with synthetic biology, Isaac received the Emerging Leaders in Biosecurity Fellowship at the Johns Hopkins Center for Health Security, which fosters the development of the next generation of leaders in biosecurity. Isaac is one of 28 individuals selected, and is also the youngest member of the 2018 class. 

iGEM delegates, including Chris Isaac (second from Left), at the BWC in Geneva, Switzerland.

“I was thrilled to have been selected as a fellow and have since travelled to Washington, DC for the first fellowship meeting. We had the opportunity to meet with staff of the National Security Council on the White House campus and discussed national-level priorities for biosecurity, challenges, and the direction that biosecurity programs are headed.”

Currently, Isaac is a Biochemistry Master’s student at the University of Lethbridge in the lab of Dr. Athan Zovoilis. In addition to graduate studies and the fellowship program, he is participating in his 7th year of iGEM, as well as continuing to develop his biosecurity software suite. 

“With both of these experiences combined, my most important takeaway is two-fold. First, there are great people working on issues of biosecurity from all sides, they come from all walks of life, and work at all levels of administration; international, national, state, provincial, academic, and non-governmental. Secondly, it reaffirms my belief that scientists need to be more involved with government, and communicate outside of academic or industrial silos. We should be seriously considering dual-use implications for even basic research that might enable other unrelated technologies to be used improperly”.

Taylor would like to thank Chris Isaac for sharing his experience.

SAMIR HAMADACHE - APRIL 13, 2018

Nucleases: “Bigger, better, stronger”

Dr. David Edgell is the Acting Chair of the Department of Biochemistry at Schulich School of Medicine and Dentistry (Western University).

While the scientific community and now much of the public is excited about CRISPR/Cas9, some synthetic biologists like Western University’s Dr. David Edgell are working away at creating the next generation of DNA-editing tools. As Associate Professor and now Acting Chair of Western’s Biochemistry Department, Edgell is engineering nucleases “to make them bigger, better, stronger…more accurate, and having a more defined function.”

Edgell’s research began with asking basic questions about mobile genetic elements—DNA fragments capable of moving from one genomic position to another. They do this by using enzymes called homing endonucleases, which are nucleases that introduce double-stranded DNA breaks at precise sequences. “It quickly became clear that we could adapt this protein for genome-editing applications,” Dr. Edgell says. “So, over the past eight years, that’s what my lab has been really moving towards.”

Specifically, the Edgell Lab focusses on developing genome-editing nucleases for applications in various model systems. His latest creation? A fusion of Cas9 with I-TevI homing endonuclease, producing a dual nuclease termed TevCas9. The easy-to-use dual nuclease offers greater target-site specificity than Cas9 on its own and circumvents one of the biggest challenges of Cas9: regeneration of the target site.

“A lot of scientists realize the limitations of Cas9”

Despite its proven utility, The Cas9 nuclease confers a disadvantage upon CRISPR-based gene-editing. When Cas9 cuts DNA, a straight cut through the two strands of DNA leaves blunt ends. This promotes a DNA repair pathway called non-homologous end-joining (NHEJ), which “will simply take those blunt ends and jam them together, regenerating the Cas9 target site,” Edgell explains. However, since this repair pathway is imperfect, bases are occasionally lost or added in the process. Eventually the target sequence is disrupted, preventing further cleavage, and effectively knocking out the targeted gene.

It’s a messy process. The length and nature of base mutations at the target site depends on the rather unpredictable cycle of target site cleavage and regeneration. Dr. Edgell’s TevCas9 dual-nuclease, consistently deletes fragments of defined length. The fragment deleted is between the cut sites of each nuclease. After NHEJ repairs the break, the target site is lost, and the futile cycle is avoided altogether.

This allows for more reliable and predictable gene knock-outs. Another crucial gene-editing task is to perform gene knock-ins by inserting new DNA fragments at targeted locations. For this, a different DNA repair pathway is needed.

In order to insert a new DNA sequence at a DNA break, repair involving homologous recombination (HR) is required. As the blunt ends produced by Cas9 promote NHEJ, HR is promoted by a staggered cut that leaves overhangs instead. This is a challenge that many scientists are currently working to overcome. “The idea is to trick Cas9 into making ends that are not blunt ends, or to add another domain onto Cas9 to promote homologous recombination in some way,” says Edgell. “It’s kind of the next big holy grail in genome engineering.”

It’s not the only challenge Cas9 currently faces. Studies in recent months have discovered a pre-existing immune response to the Cas9 protein in adult humans. Although this presents an obstacle in gene therapy efforts, Edgell is optimistic that it’s a challenge that can be circumvented because Cas9 could be engineered to make it unrecognizable to the immune system. This would be done by modifying the epitope (3-dimensional surface structure) that antibodies are recognizing.

Advancing synthetic biology at Western University

In addition to engineering more useful nucleases, Dr. Edgell is collaborating with other scientists to apply nucleases like TevCas9 for synthetic biology applications.

One project with fellow biochemistry professors Dr. Greg Gloor and Dr. Bogumil Karas is aimed at using TevCas9 as a “molecular warhead” for high-precision control of microbiome populations. In effect, harmful bacteria can be targeted without damaging helpful bacterial populations. In addition to human health applications, this approach could be extended assist in industrial food production (such as yogurt probiotics) and environmental cleanups.

In another collaboration with Dr. Karas, Edgell is working to increase the utility of the algae P. tricornutum. This species of algae is a popular candidate for biofuel production, but Karas and Edgell think it is also very promising for the biosynthesis of other high-value products.

Outside of the lab, Edgell is working with other Faculty and Administrators to bring synthetic biology to the forefront at Western University. “I think there’s a lot of interest in synthetic biology at Western. [We] are trying to develop an umbrella structure to promote synthetic biology research.” Along with Dr. Kathleen Hill, Dr. Karas, and others, Edgell has applied for a large internal grant to formalize ongoing efforts such as an undergraduate synthetic biology module, a collaborative graduate program, and the annual summer Synthetic Biology Symposium.

“This is a really exciting time to be involved in synthetic biology. The promise of synthetic biology is massive.”

-Samir would like to acknowledge Rachel Boyd’s influential help editing this article.