What were the safety issues in our project?

Our project is divided into two parts. The first part involved identifying unique tardigrade proteins that are likely part of the of tardigrades resistance to desiccation and then cloning them into E. coli to see if this resistance can be transferred to other organisms. We did not extract tardigrade DNA. All genes were synthesized from information found in public databases. This part is very straightforward and presents typical risk for iGEM projects. The bacterial chassis used was E. coli K12 derivative DH5alpha and all the parts used present no risk to humans. Since the parts and the chassis are from Group 1 organisms there were no unusual safety issues posed by this part of the project. Use of good microbiological practices would be sufficient to minimize risk.

For the second part of our project where we attempted to knocking out genes in tardigrades, we acquired a culture of the H. dujardini tardigrade strain from Carolina Biological Supply. We used microinjection to effect germline modification. Although tardigrades pose no risk to humans and are ubiquitous in the environment we still used standard lab practices for containment and treated all specimens as potential environmental contaminants. We were particularly careful about discarding tardigrade samples since they live in freshwater and are hardy, and it was not clear to us if the species used was native to our geographic area. All samples were put in the biohazardous waste after use.

Special Considerations for the CRISPR / Cas9 system

Our design incorporated additional controls used to minimise risk when using the CRISPR / Cas9 gene modification technique. Since we were altering the germline of an organism, one of our primary concerns when considering our experimental design using this system was the possibility of persistence of CRISPR DNA or even unintentionally introducing a gene drive. To eliminate this and minimize risk, we chose to design our experiment in such a way that we did not introduce DNA coding for either the Cas9 enzyme or the guide RNA into the cells of tardigrades. Instead, we chose to microinject ribonucleoprotein particles consisting of the Cas9 enzyme provided by New England BioLabs Inc. and complexed with guide RNA synthesized using the EnGen sgRNA Synthesis kit also from NEB.