Session 3

Claire Velikonja

McMaster University

Simplifying virus titre measurement via live-cell imaging: A critical step in enabling advancements in biotherapeutic manufacturing

Virus therapeutics are a promising avenue of treatment for genetic diseases, with the approval of therapeutics (e.g., Imglyic and Zolgensma) motivating researchers to translate from the bench to the clinic. However, scale-up production of virus therapeutics, such as adenovirus, is difficult due to current analytical methods available for assessing virus infectivity titer. For adenovirus, the standard for assessing titer, hexon staining, requires a minimum of 48 hours and timed antibody washes, reducing sample processing potential. Moreover, the assay relies on manual counting of the stained regions, introducing human error that can create large standard deviations. Here, we demonstrate a virus infectivity assay that leverages the Sartorius Incucyte Live-Cell Analysis System to reduce required manpower and increase assay throughput. Adenovirus with a GFP-transgene was used with the Incucyte system’s built-in segmentation algorithms to estimate virus titer by counting the appearance of fluorescently labelled cells, and the estimates were compared to traditional hexon staining. Furthermore, a statistical workflow was proposed for determining the virus titer of the sample from its dilution series, which is a persistent problem within traditional hexon staining. Overall, as virus-based therapeutics are developed, similar effort must be matched to ensure that their clinical production can be achieved. The assay in this work was developed to enable virus production and process development research, by reducing labour and time required by virus infectivity assays.

Ethan Agena

University of Toronto

Developing Genetic Engineering Tools To Expand the Product Spectrum of Anaerobic Chain Elongating Bacteria

Developing the anaerobic chain elongating bacterium, Clostridium kluyveri, as a production platform is a novel opportunity to expand the product spectrum of anaerobic bioprocesses for tandem waste utilization and chemical production. C. kluyveri can be used within microbial communities to convert industrial, agricultural, and municipal waste streams to medium-chain fatty acids (MCFAs). MCFAs are valuable platform compounds in several manufacturing sectors and can be further derivatized into fuel compounds. However, metabolic engineering of C. kluyveri for improved production of MCFAs and other compounds has not been explored as genetic tools for the microbe are lacking. The foremost barrier preventing the engineering of C. kluyveri is its native restriction-modification (RM) system which hinders the DNA uptake necessary for metabolic engineering. The current study aims to improve DNA uptake by using DNA methylation to protect plasmids from C. kluyveri’s RM system. Here, we use the Restriction Enzyme Database (REBASE) and Single Molecule, Real-Time (SMRT) sequencing to identify key DNA methyltransferases in C. kluveryi’s RM system. These DNA methyltransferases were expressed in Escherichia coli for activity confirmation and are used in vivo to properly methylate the plasmids to be delivered into C. kluyveri. This provides a unique prospect to express heterologous pathways in C. kluyveri that could enable tighter control of MCFA chain length and conversion MCFAs through heterologous pathways. This tool development is a critical step to expand potential applications of C. kluyveri and other chain elongating bacteria for anaerobic bioprocessing, which otherwise could not be achieved without metabolic engineering.

Mitchell Zak

University of Toronto

Biosorption selectivity of rare earth elements onto Euglena mutabilis suspensions and biofilms

The increasing demand for electronics has led to a desire to recover rare earth elements (REEs) from non-conventional sources, including mining and waste effluents. Biosorption is a promising method for adsorbing REEs onto microalgae, but biomass immobilization and light delivery challenges remain. Algal biofilms have been recently found by our group to solve these issues and to adsorb more REEs than suspended biomass due to the extracellular polymeric substance (EPS) matrix. However, the selectivity of REE adsorption onto algal biofilms has not been assessed alongside the role of the EPS matrix on selectivity. The maximum adsorption capacities of Euglena mutabilis suspensions and biofilms were determined for a mixed REE sulphate solution at an equimolar starting concentration. Preferential adsorption of Sm and Eu was found for both suspensions and biofilms. In addition, biofilms were also found to preferentially adsorb Yb and Lu compared with suspended Euglena. The impact of Ca2+, Mg2+, and Fe2+ on REE adsorption was also assessed. The amount of REEs adsorbed onto suspensions was found to increase when Ca2+ and Mg2+ were added to the system and there was no effect from adding Fe2+. Biofilms however had no change in the total amount of REEs adsorbed when Ca2+ and Mg2+ were added into the system whereas the presence of Fe2+ reduced the adsorption capacity by up to 25%. This is believed to be because Fe2+ competes with REE ions for binding sites whereas Ca2+ and Mg2+ do not form strong enough bonds compared to compete with REEs.

Kurt Loedige 

Western University

Surveying the Diversity of GIY-YIG Nuclease Domains for Gene Editing

Gene editing involves making specific modifications to an organism's genetic material by introducing targeted double-stranded breaks in DNA using endonucleases. The CRISPR/Cas9 system is most used for this purpose, and GIY-YIG homing endonucleases can be combined with Cas9 to create a dual-cutting targeted nuclease. TevCas9 is a dual endonuclease that includes the nuclease and linker domains of the I-TevI GIY-YIG homing endonuclease and Cas9, which allows for the specificity and programmability of the RNA-guided CRISPR/Cas9 system to be harnessed for cutting target DNA twice, resulting in discrete-length excisions. While the preference of the I-TevI nuclease domain for cleavage sites resembling its cognate motif (5’-CN↑NN↓G-3’) is a key component of TevCas9's targeting and specific activity, it can also limit the diversity of target sequences accessible to editing. To address this limitation, we are investigating the diversity of cleavage motif preferences within the GIY-YIG nuclease domain superfamily to identify domains that can be combined with Cas9 (GIY-Cas9s) and have different preferences compared to the I-TevI nuclease domain. Preliminary activity screening of uncharacterized nuclease domains has identified 17 domains that exhibit strong cleavage activity on a randomized DNA substrate library in vitro, and five of the most active domains have been further investigated and found to have diverse cleavage site preferences. This research aims to expand the versatility of GIY-Cas9 dual endonucleases as genome-editing tools and enable editing of target gene sequences that were previously inaccessible using TevCas9.