Before beginning any experimental operations, all team members completed project-specific biosafety training. The training covered essential laboratory safety topics, including the handling of experimental materials, standard biosafety protocols, prevention of misuse or accidental leakage of biological materials, and the correct procedures for emergency response. Special attention was paid to the safe handling of biological components associated with the Hepzero system—such as dCas proteins, sgRNA expression plasmids, HBV-related nucleic acid fragments, and plasmids or sequences ordered from Sangon Biotech. This ensured that every team member could confidently and safely manage both existing and newly introduced materials.
To maintain a high level of biosafety oversight, the team worked under the supervision of a designated iGEM mentor and a Principal Investigator (PI), who jointly provided continuous risk management and safety support throughout the project. The mentor and PI regularly reviewed the safety aspects of Hepzero’s experimental protocols, focusing particularly on potential off-target effects of sgRNA–dCas complexes, the biosafety of HBV-derived fragments, and risks associated with newly synthesized plasmids. They also provided ongoing optimization feedback and on-site supervision during laboratory procedures to ensure that all experiments were conducted safely and in compliance with institutional and iGEM standards.
In parallel, the team introduced a dynamic safety update mechanism to keep internal practices aligned with current biosafety standards. A safety officer was assigned to monitor updates to iGEM’s official biosafety requirements and the HBV experimental safety guidelines issued by the International Liver Transplant Society (ILTS). Relevant updates were promptly communicated and implemented, ensuring that the team’s work consistently met the highest safety and ethical standards.
From the project’s design stage, ethical and biosafety considerations were prioritized. Since the Hepzerosystem focuses exclusively on in-vitro bacterial research, risks such as viral cross-species transmission and ethical issues related to HBV-infected animal or human models were preemptively eliminated. After comprehensive evaluation of safety and feasibility, the team determined that optimizing the system in a controlled in-vitro environment would be the most responsible and secure approach—fully in line with the principle of “safety first, ethics foremost.”
Given that the HEPGERO system involves CRISPR-based gene regulation, HBV treatment targets, and newly designed genetic constructs, robust data security measures were implemented. To prevent unauthorized access or misuse of sensitive information, the team established a hierarchical information management system. When communicating research outcomes externally, potentially sensitive data—such as sgRNA “kissing-loop” sequences, dCas protein engineering details, cccDNA target sites, and key plasmid sequences—were desensitized. Instead, external communications emphasized the system’s safety features, including its non-cutting design, low off-target potential, and strict bacterial containment procedures. This strategy ensures responsible scientific communication that remains transparent while safeguarding critical technical information.
The Hepzero project operates under a comprehensive regulatory and ethical framework that integrates international and national biosafety standards. These include the official iGEM biosafety guidelines, the Biosafety Law of the People’s Republic of China, and the Regulations on the Biosafety Management of Pathogenic Microorganism Laboratories relevant to HBV-related work. Ethical principles governing the responsible application of CRISPR technologies were also strictly observed.
This framework serves as the foundation for all aspects of risk management—from pre-experimental risk assessments (such as sgRNA off-target prediction and evaluation of HBV fragment containment) to containment procedures during experiments (e.g., the use of biosafety cabinets and sterilization routines), and post-experimental reviews (e.g., validation of off-target detection and biohazard waste tracing). Through systematic training, expert oversight, and strict compliance monitoring, the team effectively managed every stage of risk control, ensuring the safety of personnel, the laboratory environment, and the surrounding community.
All biological materials involved in the Hepzero system experiments have undergone stringent safety assessments. The specific list and characteristics are as follows:
| Material Category | Specific Contents | Safety Characteristics |
|---|---|---|
| Bacterial Strains & Plasmids |
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| Engineered Nucleic Acids & Proteins |
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All plasmid construction for the Hepzero system was performed in a BSL-2 laboratory under controlled biosafety conditions. During plasmid cloning, Type IIS restriction enzymes (BsaI, BbsI) were used for digestion, and all reactions were carried out inside a biosafety cabinet to prevent aerosol formation and cross-contamination. When constructing the sgRNA library, DNA fragments containing random 3′ extension sequences were synthesized by Sangon Biotech. Upon arrival, these fragments were dissolved and inspected within the biosafety cabinet to ensure quality and prevent contamination. Plasmids ordered from Sangon Biotech were also opened and verified inside the biosafety cabinet to confirm integrity and consistency with the order specifications. Any deviations were promptly reported to the supplier. Transformation of plasmids into E. coli was conducted using the heat-shock method in sealed tubes, and subsequent plating was performed within the biosafety cabinet. Petri dishes were inverted during incubation at 37°C to minimize contamination and prevent spillage.
Positive clones were cultured in LB medium supplemented with antibiotics at safe working concentrations: Chloramphenicol (25 μg/mL), Ampicillin (100 μg/mL), or Kanamycin (50 μg/mL). Concentrations were carefully controlled to avoid promoting antibiotic resistance. Plasmid extraction was performed using commercial kits, and all waste liquids (bacterial lysates, wash buffers, and extraction residues) were collected in designated containers and sterilized before disposal through certified hazardous waste management services. Extracted plasmids were analyzed for purity and concentration using Nanodrop and agarose gel electrophoresis, with all testing conducted under sealed conditions. Gels and consumables used in the process were sterilized by autoclaving before disposal.
Operations involving engineered nucleic acids and proteins must adhere to strict biosafety protocols to ensure safe handling, prevent contamination, and avoid exposure risks.
In-vitro transcription and purification of sgRNAs:
If in-vitro synthesis of sgRNAs is required, the entire transcription process must be carried out inside a biosafety cabinet to prevent aerosol diffusion and RNase contamination. The resulting transcription products are purified using a standard RNA purification kit. Purified sgRNAs should be stored at -80 °C to minimize degradation caused by repeated freeze–thaw cycles. During all operations, disposable gloves and masks must be worn to protect both the samples from RNases and personnel from accidental contact.
Expression and purification of dCas proteins:
The expression of dCas proteins is performed through a prokaryotic expression system using the aforementioned E. coli strains. All bacterial cultivation and expression processes must take place in a BSL-2 laboratory. Following expression, proteins are purified via affinity chromatography columns. The purity and molecular weight of the obtained proteins are confirmed by SDS–PAGE analysis. After verification, used gels and consumables should be treated as biohazard waste and autoclaved accordingly. Purified proteins are stored at -80 °C, and all storage containers must be clearly labeled with the protein name, concentration, preparation date, and appropriate safety warnings (e.g., “No DNA cleavage activity. Avoid repeated freeze–thaw cycles”).
In the event of laboratory accidents, immediate containment, disinfection, and reporting are essential.
Spills of bacterial culture or samples:
Promptly cover the spill with absorbent material, then slowly pour an appropriate disinfectant (e.g., 1% sodium hypochlorite or 75% ethanol) from the outside inward. Allow at least 30 minutes for disinfection before cleaning. All cleanup materials must be autoclaved as biohazard waste. The incident should be reported to the laboratory safety officer and properly documented.
Plasmid or nucleic acid sample contamination:
Disinfect the affected area immediately. In cases involving HBV-related nucleic acid fragments, use disinfectants proven effective against nucleic acids, such as those containing quaternary ammonium compounds or hydrogen peroxide. All consumables that may have come into contact with the contaminated material must be sterilized prior to disposal.
Sequencing and handling of plasmid vectors for the Hepzero system follow iGEM’s public material repository standards, with specific correspondences defined below.
All plasmid sequencing work is outsourced to Sangon Biotech. When preparing samples for shipment, they must be sealed in centrifuge tubes, properly packaged, and labeled with the sample name and biosafety level (e.g., “BSL-2, non-infectious plasmid”). A concise sample safety description should accompany the shipment to inform the courier and sequencing company of the sample’s nature, thereby minimizing any potential biosafety risks during transport and sequencing.
Proper management of laboratory equipment and waste is critical to maintaining biosafety and ensuring the reliability of experimental data. The following protocols describe the cleaning, handling, and disposal procedures specific to the Hepzero system.
Specialized equipment used in Hepzero experiments requires dedicated cleaning and maintenance routines to prevent contamination and ensure consistent performance.
Flow cytometry:
After each bacterial sample analysis, flush the sample tubing thoroughly with a dedicated cleaning solution to eliminate any residual bacterial material and prevent cross-contamination between samples. A complete disinfection procedure must be performed monthly. Record the date and condition of the equipment after each disinfection cycle for traceability.
Fluorescence microscope:
Following observation of bacterial samples, clean the microscope stage and objectives with 75% ethanol to remove any residual material. When using oil immersion objectives, carefully wipe the lens with lens paper moistened with lens cleaning solution to remove immersion oil residues that could interfere with subsequent imaging sessions.
Reusable laboratory equipment involved in Hepzero system experiments, such as Petri dishes, centrifuge tubes, and pipettes, must be treated according to the following procedures after use:
Given the specific experimental nature of the Hepzero system, additional waste management protocols are established to ensure comprehensive and compliant disposal of all laboratory byproducts.
Bacteria-related biohazard waste:
This category includes bacterial culture media, contaminated pipette tips or centrifuge tubes, bacterial lysates, and other materials in contact with bacterial cultures. Such waste must be collected in red biohazard bags, sealed, and clearly labeled with “Bacteria-Related Waste” and the generation date. The bags are then autoclaved at 121 °C and 103.4 kPa for 30 minutes before being transferred to a licensed hazardous waste treatment company for final disposal.
CRISPR-related reagent waste:
This includes chromatography column waste liquids from dCas protein purification, residual reagents from sgRNA in-vitro transcription, and waste liquids from restriction enzyme reactions. These materials must be collected separately in designated waste containers containing appropriate neutralizing agents (e.g., protease inhibitors for protease-containing waste; heat inactivation or nucleic acid degradation reagents for nucleic acid-containing waste). Each container should be clearly labeled as “CRISPR Reagent Waste Liquid” and periodically handed over to a certified hazardous waste disposal company.
Through the application of these specialized waste management protocols—integrated with standard biosafety procedures—the Hepzero team ensures rigorous control over all biological risks associated with the project. These practices maintain a safe, compliant, and environmentally responsible laboratory environment for ongoing system research and development.
To ensure that the Hepzero project continues to operate under the highest biosafety standards as it advances, the team has established a forward-looking plan for continuous safety improvement and preparedness.
If future work involves scaling up experiments or conducting more complex bacterial operations, all laboratory facilities and equipment must be re-evaluated and, if necessary, upgraded in advance. This ensures full compliance with biosafety level requirements and prevents potential hazards associated with increased experimental complexity or volume.
The team will actively monitor updates in relevant biosafety and biotechnology regulations, both domestic and international. Whenever new standards or procedures are introduced, all members will undergo timely retraining to maintain up-to-date operational skills and safety awareness consistent with the latest guidelines.
A comprehensive laboratory safety emergency response plan will be finalized and continuously refined. Detailed countermeasures will be developed for possible incidents such as bacterial leaks or plasmid contamination. Regular emergency drills will be organized to strengthen the team’s ability to respond effectively and swiftly in real situations, ensuring that all members can act confidently and in coordination to minimize risk and maintain a safe research environment.