Engineering the Future of Gene-Editing Proteins
SAMIR HAMADACHE - APRIL 13, 2018
Nucleases: “Bigger, better, stronger”
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.