safety is important:

While working in a wet lab, one must take precautions to avoid exposure to harmful chemicals and dangerous equipment. Therefore, before proceeding to the bench, an individual must be wearing a lab coat, eye protection, long pants, closed-toe shoes, and nitrile gloves as personal protective equipment. It is also important to tie long hair back and remove loose jewelry to avoid the risk of contamination. Another layer of contamination avoidance is the use of 70% ethanol, which is effective in reducing the likelihood of microorganisms pollution. 70% ethanol is regularly sprayed on work surfaces, gloves, pipettes, and gel-casting trays.

Protocols:

Apart from personal protective equipment, every person working in the laboratory must complete a WHMIS course and laboratory safety training.

Safety in our Project:

Our team used Escherichia coli K-12 derived strains because they are not pathogenic for healthy adults as they lack the necessary machinery to colonize the human digestive tract [1].

One of the most important safety considerations we have implemented in our project is the use of Cas13a, which allows us to specifically target harmful toxins created by specific types of cyanobacteria. Cas13a’s nuclease activity is activated upon perfect base-pairing between the Cas13a guide RNA and our target molecule, mycrostin mRNA. Cas13a activation results in the cleavage of the target RNA and is coupled to trans-nuclease activity [2][3][4]. This allows us to achieve our goal of managing harmful cyanobacterial blooms and the toxins they produce without causing any off-target effects.

We are also ensuring that the theoretical final product of this project will not involve the release of any live organisms into the environment, as our theoretical final product will be a phage-like particle containing Cas13a, which involves no release of E. coli into the environment. There is little to no danger in the enclosed Cas13a causing any modifications to DNA in the environment, as Cas13a only targets RNA and does not edit genomes.

REFERENCES

Final Risk Assessment of Escherichia coli K-12 Derivatives. US EPA.

A versatile toolkit for CRISPR-Cas13-based RNA manipulation in Drosophila. Genome Biology. Doi: 10.1186/s13059-020-02193-y

Molecular Mechanisms of RNA Targeting by Cas13-containing Type VI CRISPR-Cas Systems. Journal of Molecular Biology. Doi: 10.1038/s41467-019-11126-8

Nalefski, E. A. et al. Kinetic analysis of Cas12a and Cas13a RNA-Guided nucleases for development of improved CRISPR-Based diagnostics. iScience 24, 102996, doi:https://doi.org/10.1016/j.isci.2021.102996 (2021)