General safety measures
As every person working with living microorganisms we had to follow a set of simple rules. First of all always wear a labcoat and gloves at any given time in the laboratory. Before starting and after finishing to work on a bench decontaminate the place with ethanol. All our waste and biowaste were treated accordingly to the legislation in our country.
Special safety needs for our project
Our project is based on the dCas9, a derived form of the very polemical CRISPR-Cas9 protein which raised safety issues concerning the “off target effects” (when the Cas9 is guided to and cuts an unwanted sequence in the genome). These effects led us to think about some ways to minimize the risks of unwanted targeting, both in the genome of E.coli and S. cerevisiae we are working with. Even though we won’t introduce Cas9 or dCas9 into a human cell we wanted to check if there was a risk of gene activation/inhibition if we got a large quantity of these proteins into our body. In our case (with dCas9), the risk is the following: if a guide RNA causes dCas9 to bind a regulatory region of gene in an unwanted fashion, it might be deadly for the organism.
What did we do to reduce the risk?
To assess these issues we designed a program that automatically blasts the guide RNA (gRNA) plus de PAM sequence (either CCn at the beginning or nGG at the end) against the genome of E. coli, S. cerevisiae and the human genome. When we had the result the program would give us the sequences that are not in the organisms. Note that we wanted to work with completely synthetic gRNA to have as little interference as possible. Future iGEM teams could use our software to read their BLAST results and to draw conclusions of the results that could maybe result in less “off-target effects”.