Safety is the number one priority

General Lab Safety

Before starting the project, our team completed the biosafety and the biosecurity trainings provided by the University of Toronto Biosafety office, and our project supervisor, Dr. Radhakrishnan Mahadevan. The training modules completed include lab access and rules, biosafety levels and equipment, emergency procedures etc.

Overall, our team adhered to the Canadian Biosafety Standards and Guidelines.[1] The biosafety of our lab was overseen by the University's Institutional Biosafety Committee, with administrative and technical support provided by the Biosafety Team in the Office of Environmental Health and Safety.[2]

Lab safety is lab fun.

Safe Project Design

All the biological materials were handled in open bench areas. Escherichia coli (E.coli) was the chassis organism used in our study, which is exempt from the requirements of the NIH Guidelines[3], as a risk group 1 organism[4] . We fused the protein binding domain of LacI (from E.coli) and the light oxygen voltage domain (LOV) of Avena sativa.

We used a CI repressor protein from Escherichia phage lambda in our switch. This sequence was obtained from a peer-reviewed article and has no indication of hazardous impact on eukaryotic cell lines.

The anti-CRISPR protein, AcrIIA4, was obtained from Listeria monocytogenes prophages (risk group 1). The protein has been expressed in both bacterial and human cell lines in literature with no measurable toxicity.

The anti-microbial resistance factors in our project were Standard Kanamycin and Chloramphenicol resistance casettes, which are commonly used in research and pose little health risk.

Our engineered system does not have any risk to humans or the environment since our Cas9 protein only targets metabolic genes like araC in E.Coli as well as reporter genes like GFP or mCherry. Standard decontamination and containment through thermal sterilization and certified waste disposal would also mitigate any potential release in the environment.

Future Considerations

In terms of future applications, temporal control of Cas9 activity through our switch could reduce off-target edits and increase the fidelity of genomic integrations through homology-directed repair, which should improve the safety of this technology. Although, from an ethical standpoint, there are several key shareholders including patients, medical practitioners and legislative and executive agencies that could be impacted by human gene editing. Thus, we conducted a systematic analysis of the considerations of each of those groups through interviews and a collaborative podcast series.