Non-pathogenic strains of E. coli such as BL21 and DH5α from Life Technologies and NEB Stable Competent E. coli were used for cloning of plasmids and expression of proteins of interest. Such strains have been modified such that the pathogenic gene islands have been removed and the strain used is considered non-pathogenic. These strains are Risk Group 1 and were handled in a BSL2 Biosafety cabinet. The mammalian human cell line, HEK293T is classified under Risk Group 2 and was also handled in a BSL2 Biosafety cabinet. Human embryonic kidney cells that have been transformed and immortalised with SV40 large antigen. Training for handling of mammalian cells or any new experimental protocol was provided by the principal investigator (PI) to all team members prior to the experiments.

In our project, we created a fusion protein (RESCUE editor) made up of dCas13b and rAPOBEC1, and a RESCUE reporter to indicate editing efficacy. Our fusion protein is designed to edit a specific RNA target from C-to-U. As the RESCUE editor targets only RNA, editing would be transient and reversible. To add on, the RESCUE editor consists of a ‘dead’ Cas13b, thus preventing cleavage of RNA strands. Our fusion protein gene was assembled together and cloned into a mammalian expression plasmid vector using competent E. coli cells. Subsequently, we transfected the fusion protein and a gRNA targeting a specific base on mRNA into the HEK293T human cell line to test the RNA editing efficacy in vivo.

Even without a gRNA, the RESCUE editor may also make non-specific edits. However, Cas13b targets RNA strands and not DNA. Therefore, any non-specific edits would not be passed on to the daughter cells. In addition, mRNAs have relatively short half-lives. Thus any lethal changes made would also be degraded quickly and will not be passed down to the next generation.

In such a case, our fusion protein may be used for targeted RNA editing. We can generate specific gRNAs to target RNA sequences and modify the RNA sequences in the body. This is also to minimise off-target editing.

The RESCUE reporter system was cloned into a mammalian expression plasmid vector using competent E. coli cells. It was subsequently transfected and tested in vivo using a fusion protein made up of Cas9 and rAPOBEC1 to test for the functionality of the reporter system.

All bacterial work, transfections and cell cultures were done in certified BSL2 biosafety cabinets.

All our team members have undergone and passed the Chemical, Biological and Fire Safety training requirements from the Office of Safety, Health and Environment (OSHE), the department in charge of Laboratory and Work Safety at the National University of Singapore. Furthermore, laboratory specific training has also been given by the respective PIs of the lab where the laboratory work was carried out (SPS teaching lab at Faculty of Science, A/P Liou Yih-Cherng’s Lab at Department of Biological Sciences, Asst. Professor Alan Prem Kumar’s Lab at Cancer Science Institute and A/P Matthew Chang’s Lab at SynCTI). Necessary Hepatitis B vaccinations were also taken in preparation for working with mammalian cell lines.

For each protocol used in our experiments, we have a separate risk assessment. By performing the risk assessment, team members will be aware of the risks involved in the experiment and consequently take appropriate safety precautions. Please refer to our ‘protocols’ page for more information. Our laboratory is equipped with biological and chemical spill kits, and all members of our iGEM Team are trained to handle Biological and Chemical Spills.

All the aforementioned labs are classified as Biosafety Level 2, according to the classification by the World Health Organisation (WHO) and the Genetic Modification Advisory Committee of the government of Singapore. The laboratory is controlled by card access which only team members or users that have passed all the safety training requirements are given. All biosafety cabinets, fume hoods and the autoclave have been recertified to be working properly by the respective companies and certified inspectors.

Bacterial work and mammalian cell culture are performed in separate BSL2 Biosafety Cabinets to minimise cross contamination, while manipulation of DNA is done on the open laboratory bench. No cytotoxic reagents are used in our laboratory; SYBR Safe and RedSafe DNA stain for visualisation of DNA is used rather than Ethidium Bromide.

Liquid biological waste is decontaminated using 10% Bleach for 20 min before disposal as normal waste, while solid biological waste are sent for incineration at a local incineration plant devoted to medical waste (Sembcorp).

For the fulfillment of requirements for safety from the iGEM foundation, we have submitted the safety forms here.