Ensuring the Safe Application of PICasSO
The PICasSO System: Identifying Possible Risks
After consultations with experts, we came to the conclusion that our project poses minimal risk. However, bringing two DNA sequences into close proximity in vivo, especially if one of them is an enhancer, poses potential risks, such as carcinogenesis and cell dysfunction. Additionally, off-target effects and the incomplete understanding of 3D genome organization make it challenging to fully assess all possible outcomes. Of note, these risks would only apply to applications in animal models or even patients and are of no danger in context of cell and tissue culture experiments.
The PICasSO System: How We Eliminate These Risks
Our system is designed primarily for research purposes, though it holds therapeutic potential. The main risks are related to handling materials at Biosafety Level 1 (BSL-1). As long as all necessary BSL-1 safety protocols are followed, our system can be safely operated without posing risks to humans.
We consulted Dr. Timo Kehl, Head of Biosafety at DKFZ Heidelberg, for expert guidance on the safety of our project. He confirmed that our work, using E. coli K-12 strains, is generally safe, provided we avoid introducing toxins and minimize off-target effects. To further reduce risks, Dr. Kehl advised us to avoid sequence homology with oncogenes or tumour suppressor genes in our guide RNA design, which helps minimize off-target activity.
Our in vitro enhancer hijacking experiments will remain at BSL-1, as long as we avoid using viral or other BSL-2 systems. Additionally, we focus on using "dead" Cas proteins, which are catalytically inactive and cannot cleave DNA strands. For constructs encoding catalytically active Cas9 and Cas12, we have utilized safe harbour loci, such as FANCF, VEGFA, and CCR5, which are commonly used in genome editing experiments.
In future applications, our system could be adapted for use with various cell lines and under different conditions, which may require higher Biosafety Levels depending on the experimental setup. For any therapeutic applications, rigorous testing would naturally be necessary to ensure safety and efficacy. By adhering to these precautions, we aim to mitigate the potential risks associated with our research, ensuring that our system is safe both for researchers and for potential future applications.
Our Delivery Platform
We placed a strong emphasis on creating a system that prioritizes safety at every step. In particular, we focused on ensuring that our novel delivery system, based on conjugation from engineered bacteria to mammalian cells, poses no risk to the environment or humans. That is why we developed our system to be solely used as an efficient and easily scalable delivery platform for our PICasSO system. It can safely be used in R&D to deliver genetic sequences to mammalian cells in vitro - something Dr. Marta Robledo, a bacterial conjugation expert, ( See our integrated human practices page ) gave us positive feedback about.
Dr. Kehl emphasized that conjugation poses low risk as long as we don't transfer harmful elements like pathogenic proteins or viral factors, and the required biosafety level would only increase if we reached DNA transfer efficiencies similar to BSL-2 systems.
It could be hypothesized that DNA delivery in a living organism via this platform might pose risks of immunogenicity due to the bacterial DNA, which would be automatically co-delivered. That is why we addressed and respectively minimized any risks which might occur. First, the bacterial strains we used to harbour the conjugative machinery and the mobilizable plasmid are safe for humans and classified as BSL1.
As our system of one helper and one mobilizable plasmid can also be placed within another bacterial strain, the Biosafety Level shall be reassessed upon each experimental set-up to ensure ultimate protection for the operators and the environment. Moreover, we conceptualized our delivery system to adhere efficiently only to certain surface markers in order to promote cell type-specificity. Even in the unlikely event that the bacteria come into contact with the human body, our system is designed to ensure they do not preferentially adhere to random mammalian cells or transfer DNA. Lastly, we seek to investigate the cell-type specific delivery of the cargo by encoding it behind a cell-type specific promoter which would provide a second level of protection of the human cells from accidental intake.
Our Lab
Safety in Our Lab
We conducted our wet lab work at the Students' Laboratory at BioQuant in Heidelberg. All work conducted in these spaces and with the available equipment was fully compliant with regulations in Germany and the European Union. We ensured that all activities adhered strictly to German national biosafety regulations. Every plasmid created and bacterial strain used was strictly documented in the Green Book and a hard copy of the book was located in the laboratory space. Furthermore, we have taken comprehensive steps to address the safety aspects of our project and implemented effective risk mitigation strategies to minimize any potential hazards.
Waste Disposal
The waste disposal system complied with all relevant German and European regulations. BSL-1 waste was collected separately from uncontaminated waste and autoclaved prior to disposal. Liquid BSL-1 waste was also collected and autoclaved accordingly. Waste management was handled by trained personnel at BioQuant, who oversaw its proper disposal.
Mammalian Cell Culture
For work with mammalian cell cultures, we utilized the BSL-1 facility at the Institute of Pharmacy and Molecular Biotechnology in Heidelberg. Before conducting any work at this facility, all involved team members completed the necessary training. Mammalian cells were handled under a Vertical Laminar Air Flow hood to protect the cells from external contaminants.
The lab is classified as BSL-1 according to the Biological Agents Ordinance in Germany. Consequently, all participants involved in wet lab work underwent the required training. The training was conducted by one of our PIs, Prof. Stephan Wölfl, and the technical assistant responsible for the lab, Nina Beil.
Attendance was mandatory for all team members, and participation was documented with signatures. During the training, we were introduced to the lab's safety features and instructed on how to respond in case of an emergency or fire. To ensure personal safety, wet lab work was only permitted if at least two team members were present in the lab at the same time.
The consumption and storage of food, drinks, makeup, cosmetics, and other non-lab items were strictly prohibited, except for hand care products approved by the University of Heidelberg for use after applying disinfecting agents. Wearing lab coats, gloves, and, when necessary, protective glasses was mandatory at all times. Additionally, only long-sleeved clothing and closed-toe shoes were permitted in the BSL-1 rooms. The laboratory was equipped with an air exchange system following the existing regulations. Team members with long hair were required to keep their hair tied back for safety.
Our Instruments and Machines
Our wet lab work required various instruments and devices - some of them inside our lab space, others in other labs. For each instrument, team members had to pass training provided by BioQuant experts before the first use. According to the in-house regulations of Heidelberg University next to every machine, there must be a manual explaining how to operate the machine, what possible risks arise from it and what precautions are to be taken.
In recent years, the only way to cut gels in the BioQuant lab was under the gel documentation station with UV light, posing a potential risk. To address this, we invested €440 from our budget in a new gel cutting station (See Figure 1), which will remain in the lab even after our project is completed. We are pleased to have improved the working conditions for ourselves and for future lab users in the long term.
Figure 1: Gel Cutting Station
Chemicals
The Heidelberg University has established the Dangerous Materials Registry Information System (DaMaRIS) in compliance with the German Hazardous Substances Ordinance and European legislation. All hazardous chemicals used in our laboratory were registered within this system.
The initial BSL-1 training also included instructions on the safe handling and proper storage of various chemical groups. A folder containing safety data sheets (SDS) for every chemical in the lab was kept in an easily accessible location, allowing team members to review potential risks and safety precautions at any time.
To further enhance safety, access to certain chemicals, such as BME and cytochalasin D, was restricted. These substances were stored in separate compartments accessible only to the subteam working with them. Additionally, we made conscious choices regarding our lab chemicals, opting to use SYBR Safe instead of Ethidium bromide for nucleic acid staining, thereby minimizing potential risks.