As our project involves the use of dcas9 and sgrna, safety considerations needed to be made while designing our project.

Safety Report

Abstract

Endonucleases, such as CRISPR/Cas9, though known for their potential in synthetic biology to result in double-stranded breaks in the DNA, allowing for efficient insertions of target mutations, harbour the danger of creating a gene drive which can affect and propagate in the wild-type strain species. We, uOttawa iGEM team, will demonstrate that despite using dCas9, our project does not consist of a gene drive.

Project description:

The goal of this project is to overcome the lack of transcription factors when designing genetic networks in yeast. We use dCas9 machinery (including gRNA) to assess the possibility of designing a NOR gate.

Guarantee of no release:

All project parts remain strictly in the laboratory. No substances are produced by our network. The successful construction of our network is be assessed by measuring fluorescence due to GFP using a flow cytometer. Undesired yeast strains are sterilized.

Safety of project:

The project is safe. Saccharomyces cerevisiae genome is modified through basic transformations.

Unlike Cas9, dCas9, which is used in this project, has deficient endonuclease activity, because the protein domain conferring endonuclease activity is deleted from the full Cas9 protein. Thus, dCas9, cannot break double-stranded DNA, an otherwise essential step in the synthesis of a gene drive. In this experiment, dCas9 is used in combination with guide RNA to bind to a promoter site to regulate the expression of gene via steric hindrance.

Below are proofs from literature:

"We have shown recently in bacterial and human cells that the endonuclease domains of the Cas9 protein can be mutated to create a programmable RNA-dependent DNA binding protein (Qi et al., 2013). Targeting of catalytically inactive Cas9 protein (dCas9) to the coding region of a gene can sterically block RNA polymerase binding or elongation, leading to dramatic suppression of transcription in bacteria."

“Here, we show that dCas9 can be used as a modular RNA-guided platform to recruit different protein effectors to DNA in a highly specific manner in human cells and the budding yeast Saccharomyces cerevisiae. We show that both repressive and activating effectors can be fused to dCas9 to repress or activate reporter gene expression respectively. We also show CRISPRi can be used for multiplexed control of endogenous genes. Using a dCas9 fusion protein, we further show that the system can be used to stably repress genes with comparable gene silencing efficiency typically achieved by RNA interference (RNAi) while minimally impacting transcription of non-targeted genes."

Source : Gilbert, Luke A. et al. Cell , Volume 154 , Issue 2 , 442 - 451

First, we integrate a gRNA responsive promoter (pGRR) which controls GFP production. Then, we insert a 9Cas9-Mxi1 plasmid in W303 yeast strain. Finally, we integrate two gRNA sequences complementary to pGRR. The production of each gRNA is controlled by synthetic inducible promoters, GAL4 and Tet-O. dCas9-Mxi1 complexes with either gRNAs and hybridizes to pGRR which suppresses the production of GFP. Overall, the project poses no health and environmental risks, given that the synthetic genetic network has no phenotypic effects on other organisms. Furthermore, no project-related material will leave the laboratory for reasons other than sterilization for discarding.

Safeguarding gene drive experiments in the laboratory

As per iGEM's Safety Committee regulations, our laboratory and team strictly adhere to the recommendations proposed by Akbari et al in “Safeguarding gene drive experiments in the laboratory”.

According to Akbari et al., our experimental design, as explained above, “[does] not risk creating a gene drive system because cassettes encoding [dCas9] and sgRNA are not inserted into the cut site or located adjacent to one another in the genome and can thus be safely used by researchers without additional precautions.” [dCas9 will be maintained as a plasmid. There is no cut site due to the lack of endonuclease activity in dCas9.] Furthermore, our transformations are conducted on a haploid strain of yeast. The yeast will not be crossed. There will be thus no risk of conversion of heterozygotes into homozygotes within the population. Finally, the study involves the introduction of genetic parts with synthetic sequences in laboratory mutant strain of yeast and is not introduced to the wild strain.

Regardless, we follow the following stringent confinement strategies:

Molecular: The components of the system are not adjacent. dCas9 is in a plasmid, while the gRNAs will be integrated directly to the yeast genome. Study is performed on a mutant strain, not a wild strain. No genetic parts are produced commercially. We use homologous recombination to construct our entire network.
Ecological: Study is performed in a sterilized and confined laboratory environment, outside the habitable range of the host organism. The area contains no potential wild mates.
Barrier: Our transformations are performed in sterilized, low-temperature environment. Because our project does not consist of a gene drive, neither does our BioBrick.

Experience with dCas9

Our lab setting has previous experience working with synthetic gRNA networks. We have undergone necessary biosafety training from the Office of Risk Management - Environmental Health & Safety (ORM). We have thus fully assessed the project and have concluded that it is safe. Lois Sowden-Plunkett, Assistant Director, Biosafety, Radiation Safety and Laser Safety, whose contact information is given below has allowed us to proceed with the project. We are also in contact with Miles Gander, expert on dCas9-involving circuits and author of "Digital logic circuits in yeast with CRISPR-dCas9 NOR gates" (DOI: 10.1038/ncomms15459) from the University of Washington.

Conclusion

Despite using dCas9 to construct a NOR gate, no gene drive is created. By following the due process, we were able to design a project that is safe for the lab team and the greater community. Furthermore, by thoroughly understanding the established safety procedures we were able to gain insight into the policy surrounding research. uOttawa iGEM has an ongoing commitment to promoting safe research environments for the students and community.

Let’s Talk Science

In an effort to extend our reach into our university and city communities, the University of Ottawa iGEM Human Practices Team partnered up with Let's Talk Science for the 2017 season. Let's Talk Science is an award-winning, national organization that creates and delivers programs to engage youth and educators in science, technology, engineering and math (STEM). As a team, we recognized that our interests as Policy and Practices overlapped with the interests of Let's Talk Science in educating youth about the possibilities that science, including the field of synthetic biology, brings. We established communication with the Ottawa Chapter of Let's Talk Science and a great partnership was established for the 2017 season.

Adventures in Science

We also had a wonderful opportunity to work with Adventures in Science, a camp run in Ottawa that teaches science to youth during the summer in fun and engaging experiments. We spoke to campers about iGEM and the wonders of genetic engineering, and performed age appropriate synthetic biology activities to spark interest.

SynBio Science Research Workshop

During the 2017 year at the University of Ottawa, we organized a Research Skills Workshop. We spoke undergraduates about the various research opportunities available to them and how to get involved, particularly in synthetic biology. The event also consisted of an opportunity to ask questions to current grad students, and hands on experience in a synthetic biology wet lab

Investigating Opinion of Genetic Engineering

In order to gain an understanding of current thoughts on genetic engineering, an investigation, through interviews and surveys, targeting a wide range of career and education levels was conducted and the results are discussed in this report.

The results of the investiation will allow for improved responsiveness to the community with regards to human practice and educational outreach.

Team:uOttawa/Human Practices

Policy and Practice