Our Approach to Biological Safety Concerns Regarding Our CRISPR Project

Identifying Safety Issues in Our CRISPR Project

Working in a lab can be fun but also dangerous if safety for the student, society, and environment are not addressed. Therefore, the safety issues of these three important entities will be examined under the scope of risk assessment. To assess risk for a biological material many factors have to be considered, which include but are limited to: pathogenicity, ability to survive outside of the lab, public awareness of the biological agent, availability of a prophylaxis, environmental impact, and ability to monitor biological material that are potentially dangerous.

We assess risk for the biological material involved in our project and its potential impact to the researchers, public society, and environment by taking into account two components of risk: probability and hazard. With probability, we ask ourselves:

• What are the likely and unlikely (including worst-case) scenarios of conducting research in our project?
• Is there a potential risk that may compromise in any way researchers, society, or the environment? How likely that risk comes to fruition?

Questions to consider about hazards associated with biological material risk are:

• If the project works, what may be the potentials hazards to researchers, society, and the environment?
• By what mechanism may the engineered organism of the project affect researchers, society, and the environment?
• If the engineered organism is used outside the lab, how may natural mutations to the organism's DNA affect and alter the functionality and concomitant risks to researchers, society, and the environment?

We assess biological safety risks with these questions in mind in an effort to make our project acceptably safe according to the standards set by organizations, such as the World Health Organization (WHO), the Convention on Biological Diversity, the National Institute of Health (NIH), American Biological Safety Association, Center for Disease Control, among other risk management groups, and conferences with an emphasis in synthetic biology and safety such as the SB5.0 conference. In addition, complying with these safety standards will make the project and its future directions more approachable for acceptance by the public society.

Researcher Safety

Ensuring the safety of the USC iGEM lab and its researchers is critical to the success of the program. We have therefore taken initiative that each member understands and is aware of the following:

• Physiological and environmental risks associated in experimenting with a certain biological material.
• Course of action to take, as standardized and/or recommended by risk management groups, if the biological material is inappropriately exposed to the researcher and/or the environment.
• Knowledge of lab equipment to utilize to reduce the possibility of inappropriate biological material exposure and protect the researcher if such exposure occurs.
• Knowledge of safety protocols and location of safety equipment in case of inappropriate exposure, including emergency situations.

The lab, in which USC iGEM works under, is certified for Biosafety Level 1. Researchers are required to wear personal protective equipment (PPE) which include goggles, gloves, and lab coats so that the eyes, hands, and body, respectively, are always protected from possible exposure to a biological material. Potentially harmful biological materials and chemicals are used daily in the lab. Below is a description of each one we encounter on a daily or limited basis as well as the measures taken to comply with USC's stringent safety standards so that we maintain the highest quality of our safety:

• Ethidium Bromide (EtBr) is an aromatic chemical agent often used for staining of DNA during agarose gel electrophoresis. It binds with DNA and fluoresces with an orange color under UV light. EtBr may be a mutagen and carcinogen since it is believed to intercalate with double-stranded DNA, therefore inhibiting the DNA from undergoing important biological processes such as replication and transcription. Special care is definitely taken when handling EtBr. Proper PPE is used, and disposal of the chemical is handled by the researchers in accordance with EtBr's Material and Safety Data Sheet (MSDS). Pipette tips exposed to EtBr are ejected to a chemical waste basket, which also include used agarose gels that contain a residual amount of EtBr. In addition, nitrile gloves are disposed of after pipetting EtBr to the agarose gel solution to prevent secondary exposure of EtBr.
• Ultraviolet (UV) light is used for imaging of stained DNA in agarose gel electrophoresis. However, unprotected and direct exposure of UV light is known to be harmful to the skin and eyes and may lead to skin cancer and eye-related problems. UV light damages DNA by multiple mechanisms. One involves the binding of two adjacent thymine nucleotides in the DNA sequence to make a thymine dimer, which results in distortion and reduced functionality of the DNA. When imaging DNA from a gel, researchers ascertain that the UV light is turned off and only turned on when the gel has been properly placed inside the imaging machine and its door closed so UV light exposure is shielded from the researcher. Similar steps are taken when taking the gel out. In gel extraction, a protective shield is worn protecting the face from UV light exposure.
• Descendants of two E. coli isolates, B and K12, have been used as tools and model organisms in our project, in particular, strains BL21(DE3) from the B isolate and DH5a and HT115 from the K12 isolate. These strains are known to be non-pathogenic and are extremely unlikely to survive outside of the lab or in human hosts, despite some conferring a variety of basic antibiotic resistances (e.g. ampicillin, kanomycin, chloramphenicol, and streptomycin). Nevertheless, prevention of contact from the bacteria is done by requiring the researchers to wear proper PPE and decontaminating all laboratory materials exposed to the bacteria with 10% bleach before disposal.

To ensure the researchers of USC iGEM are acceptably safe from physical harm by biological material or chemical exposure, each team member attended a Safety Orientation Course conducted by USC's Career and Protective Services (CAPS). In addition, the protocols used in our project have been approved by the Institutional Biosafety Committee.

Public Safety

Our concern for public safety with respect to our project can be categorized as follows:

• How may our immediate surroundings, which include the university campus and its neighboring communities, be affected by our project?
• How may Greater Los Angeles as well as the general public be affected by our project?
• What nonphysical harms are associated with the project? (Note: We are not considering physical harms, here. Those are addressed in the previous two questions.)

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Environmental safety

The safety of the surrounding environment should not be compromised. The E. coli strain is non-pathogenic and are disinfected with the proper chemicals when cleaning materials.

BioBrick Safety Issues

No, our BioBrick parts and novel ones do not pose any issues in safety. All parts are being transformed in non-pathogenic E. coli and being expressed in vivo.

Biological Safety Provisions

Yes, the safety aspect of our project is being supervised by the Institutional Biosafety Committee at USC. All of the protocols are approved by the IBC at USC.

Thoughts on How to Improve Safety in iGEM

Sometimes, there seems to be a disconnection between the scientific community and the general public when discussing safety issues that stem from scientific research. With a new and rapidly evolving field that is synthetic biology entering the frame, we encourage our fellow colleagues and teams to be open-minded and communicative to the public regarding not only the benefits of the projects and synthetic biology as a whole, but also the potential harms, both physical and non-physical (e.g. morality) so that the public can be well-informed and hopefully accepting of this new science.