Safety

Our project fully complies with iGEM’s and NTU’s safety regulations, including chemical safety, biological safety, and data protection.

Our Lab

We work in a Biosafety Level 2 laboratory that is equipped with the appropriate safety features for our project, such as a biosafety cabinets, a chemical fumehood, chemical storage cabinets, an eyewash station and a shower.

Project Risks

Non-White List Organisms and Parts

Saccharomyces cerevisiae (Yeast)

Yeast is a type of spore-forming fungi and is classified as a Risk Group 1 (https://responsibility.igem.org/guidance/risk-groups) organism. Our team has reviewed the risks of using this organism for our project by submitting a Check In Form, and is also summarised below:

• Hazard: Individuals may be allergic to S. cerevisiae, leading to skin rashes, respiratory problems, or other allergic reactions upon exposure.
  Precaution: In order to mitigate allergies caused by accidental exposure, all members working with S. cerevisiae will be provided with appropriate personal protective equipment such as lab coats and gloves.

• Hazard: S. cerevisiae, can contaminate other cell cultures upon exposure.
  Precaution: Aseptic techniques will be followed strictly to minimise cross-contamination with other samples.

• Hazard: Working with S. cerevisiae will require the use of centrifugation machines, thermocycler, and autoclave machines, which could explode if not handled properly.
  Precaution: All user manuals and safety protocols will be read thoroughly and followed strictly while using machines in the laboratory.

Prime Editing Guide RNAs (pegRNAs)

Our team uses pegRNAs that target human genes such as HEK3 and RNF in an attempt to showcase the editing efficiencies of our designed reverse transcriptases. As they target human genes, the parts will require Check In prior to usage. Our team has reviewed the risks of using these parts for our project by submitting a Check In Form, and is also summarised below:

• Hazard: The pegRNA by itself is not hazardous but when combined with the Prime Editor (PE), may induce unintended changes in the genome.
  Precaution: Plasmids containing pegRNA and PE are stored separately. It is ensured that these plasmids are only mixed right before transfection of the cells, which are discarded in 72 hours, after analysis. Proper lab aseptic techniques are carried out as well.

CRISPR Associated Protein 9 (Cas9)

Our team uses Cas9 which creates single-stranded nicks at the target sites. The organism of origin for Cas9 is Streptococcus pyogenes serotype M1 which is classified under Risk Group 1 (https://responsibility.igem.org/guidance/risk-groups). Hence, the parts will require Check In prior to usage. Our team has reviewed the risks of using these parts for our project by submitting a Check In Form, and is also summarised below:

• Hazard: Cas9 can potentially result in off-target genome effects. Cas9 technology, which includes the development of the PE and pegRNA, can induce site-specific DNA mutations in human DNA.
  Precaution: Plasmids containing pegRNA and PE are stored separately. It is ensured that these plasmids are only mixed right before transfection of the cells, which are discarded in 72 hours, after analysis. Proper lab aseptic techniques are carried out as well.

PE3b Nicking Guide RNAs (ngRNAs)

Our team uses ngRNAs which nick the non-edited strand after the pegRNA-targeted strand is edited. As they target human genes, the parts will require Check In prior to usage. Our team has reviewed the risks of using these parts for our project by submitting a Check In Form, and is also summarised below:

• Hazard: ngRNAs have spacers that specify the target site in the human genome. The ngRNA by itself is not a hazard but when combined with the PE, may induce unintended changes in the human genome.
  Precaution: Plasmids containing pegRNA, ngRNA and the PE are stored separately. It is ensured that these plasmids are only mixed right before transfection of the cells, which are discarded in 72 hours, after analysis. Proper lab aseptic techniques are carried out as well.

Reverse Transcriptases (RTs)

Our team uses RTs which is a type of DNA polymerase that transcribes single stranded RNA into complementary DNA. The organism of origin for the RTs are Moloney Murine Leukemia Virus (MMLV) and CD Bovine Leukemia Virus (BLV) that are classified under Risk Group 2 (https://responsibility.igem.org/guidance/risk-groups). Hence, the parts will require Check In prior to usage. Our team has reviewed the risks of using these parts for our project by submitting a Check In Form, and is also summarised below:

• Hazard: BLV and MMLV are both types of retroviruses. Hence there is a risk of exposure to these infectious agents without proper precautions being taken.
  Precaution: Our lab will not be working with any of these viruses. The PE plasmids containing the RT will be commercially obtained from Addgene (e.g. plasmid #132775)

Nature of Project

As our project is foundational in nature, the application of it in the real world is highly limited at present. There are no immediate risks associated with the use of our project, but this might change in the future when prime editing starts to see actual applications in various areas.

Hazardous Chemicals

As with any biological project, chemicals will be used. Some of the chemicals to be used and their associated risks include:
• Ethanol (Flammable)
• DMSO (Flammable)
• Extraction kit buffers (irritants)
To mitigate against accidental exposure to such chemicals, basic personal protective equipment (PPE) will be worn and proper storage and handling of them will be learnt prior to usage.

Security Risks

Collection of personal data such as name and email address will be inevitable as registration is required for the synthetic biology workshop that will be hosted by our team. As part of Singapore’s Personal Data Protection Act 2012, all sensitive information will be kept confidential. Consent will also be requested from the relevant parties with regards to the use of their personal data.

Managing Risks

NTU Occupational Safety and Health (OHS) Management

NTU OHS has mandated that all students involved in research are to complete various risk assessment modules before embarking on their projects. This includes modules for chemical safety, biological safety and data protection. The specific list of modules completed by our members prior to the start of the project is shown below:
1. Safety Induction
2. Basic Safety Training
3. Understanding Signage
4. Risk Management
5. Biological Safety Training
6. Chemical Safety Training
7. Donning a disposable respirator
8. Fitting a half-mask respirator
9. Personal Data Protection Act (PDPA) e-Learning Programme
Via these modules, our members are taught the explicit instructions pertaining to biosafety and biosecurity practices, containment levels, risk assessments, and the proper handling of hazardous substances. These instructions ensure the safe and responsible execution of work. By adhering to these regulations and following the provided guidance, our project can be conducted under controlled and safe conditions. For more information regarding our team's dedication to safety, check out our submitted Safety Form.