A recurring theme in our public engagement experiences was that the use of CRSIPR to engineer organisms was a general concern, particularly when the CRSIPR system itself was perpetuated. We wanted to respond to this concern by making our use of CRISPR as contained as possible. This was one of the main reasons we took extra care and decided not to use the Cas9 gene but the Cas9 protein itself. In our experiments we microinjected the tardigrades with a ribonucleoprotein particle (the Cas9 protein complexed with guide RNA) rather than a plasmid expressing Cas9 and a guide RNA. This ensures that the system is transitory since both the protein and guide RNA will degrade, and also will not be passed on in the tardigrade progeny. We also were very careful to contain the tardigrades by treating them as we would bacterial cultures and discarding them in the biohazardous waste. Although they are not pathogenic, they could be an invasive species if this particular species of tardigrades is not native to the area, and certainly if we genetically modify them they should not be released into the environment under any circumstances. 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.

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