In this study, we employed synthetic biology approaches to assemble biological parts, constructing recombinant plasmids for Cas12a expression and transforming them into E. coli competent cells for protein production. Leveraging the expressed Cas12a protein combined with RPA technology, we established an RPA-Cas12a detection platform based on Cas12a trans-cleavage activity. This platform enables dual-output diagnosis (fluorescence/lateral flow) of Dystrophic Epidermolysis Bullosa (DEB) within <90 minutes. Prior to experiments, all members completed iGEM safety training (aseptic culturing, emergency response) to ensure zero incidents. To address ethical and biosafety concerns from engineered components (e.g., Cas12a-crRNA complexes, amplified DNA), stringent protocols were implemented: ethanol/thermal deactivation of nucleic acids, BSL-2 containment for bioactive materials (sealed systems for lysis/centrifugation), operations in dedicated biosafety cabinets, and post-use autoclaving. All instruments required certified operator training, with specialized equipment (e.g., autoclaves) handled by licensed professionals. Emergency response personnel and measures were maintained throughout.

Prior to wet lab work, all members completed institution-certified bio-safety training covering:

Core protocols: Bio-safety levels (BSL-1/2 distinctions), good microbial techniques (aseptic plating), and bio-safety equipment operation (Class II cabinet validation);

Containment measures: Sterilization/disinfection (autoclave protocols), biological waste segregation, and physical/personnel bio-security;

Emergency readiness: Chemical spill response, electrical/fire safety, and transport rules for engineered strains.
Training included competency assessments supervised by teacher, our faculty bio-safety officer. Experiments were conducted under direct oversight of certified experts, with strict adherence to instrument-specific protocols.

Prior to experimentation, comprehensive safety training was conducted for all equipment, instruments, and reagents. For specialized equipment (e.g., autoclaves, high-speed centrifugal), operation was restricted to certified personnel who performed pre-use safety checks and usage logging. Laboratories housing such equipment required faculty or staff supervision, prohibiting student solo access. Instrument training emphasized risk mitigation: thermal gloves were mandated for heating devices (water baths, PCR machines, microwaves) and cryogenic exposure (-80°C freezers, dry ice); centrifuges required strict load balancing and lid sealing; and shaking incubators displayed "Caution: Pinch Point" warnings. All electrical circuits were configured compliantly, isolated from water/fire hazards. Reagent selection prioritized low-toxicity/non-toxic alternatives, with PPE inspections (lab coats, gloves, masks) pre-experiment. During agarose/SDS-PAGE gel preparation, novel low-toxicity nucleic acid dyes/coagulants were used, supplemented by training for future dye-handling scenarios. Commercial low-irritant stains and deionized water were employed for SDS-PAGE staining/destaining within fume hoods to prevent inhalation exposure. Microbial experiments were confined to biosafety cabinets with gloved handling. Although other reagents posed minimal hazards, glove usage was universally enforced during reagent contact.

Prior to conducting any experimental work, our team underwent safety training under the guidance of our faculty advisors, covering fundamental laboratory protocols and proper handling of biological materials. All experiments were performed under the direct supervision of at least one qualified instructor to ensure compliance with safety standards. We established dedicated work zones for specific procedures, including a designated biosafety cabinet for Cas12a protein handling and a separate electrophoresis area for nucleic acid analysis with nucleic acid dyes and coagulants containment measures. Strict laboratory rules were enforced, including a complete ban on food and drinks, mandatory use of personal protective equipment (lab coats, gloves). After experimental session, we implemented thorough decontamination procedures, with all work surfaces disinfected and biological waste properly segregated and autoclaved. The team also completed emergency training, to ensure readiness for potential laboratory incidents.

Bio-safety Risk and Measures：Our project involves handling fetal cfDNA and CRISPR-Cas12a components, posing potential bio-safety risks including sample contamination and unintended nucleic acid exposure. To mitigate these risks, we implemented strict containment measures: all procedures were conducted in BSL-2 facilities with PPE (gloves, lab coats), and work surfaces were routinely decontaminated with bleach and UV treatment. Synthetic crRNA and Cas12a protein were strictly used in vitro, with no live GMOs involved. Biological waste (RPA products, DNA extracts) was autoclaved before disposal, while hazardous chemicals were segregated for certified disposal. These protocols minimized environmental and personnel risks while maintaining experimental integrity.

All experiments strictly adhered to institutional bioethical guidelines, utilizing only Risk Group 1 chassis organisms (E. coli BL21/DH5α) with no human/animal testing involved. Synthetic components (crRNA, Cas12a) were confined to in vitro systems under IRB oversight (#EB-2025-ETHICS), ensuring zero ethical concerns.

The safety of the detection platform and products constructed PrenatalEB-Detect project embeds ethical rigor by mandating genetic counseling to prevent misinterpretation of results, with all EB pathogenic gene sequences sourced exclusively from NCBI databases to ensure traceability. Patient privacy is protected through HIPAA-compliant encryption, while cost optimization (<$15/test) and culturally-sensitive reporting promote accessibility. Post-detection samples undergo complete biohazard deactivation: blood cells are chemically lysed (rendering pathogens non-infectious), and lateral flow strips contain only immobilized gold-labeled antibodies/protein markers—posing zero biological risk. Residual DNA/proteins are rapidly degraded via household disinfectant spray (10 min contact time), ensuring non-toxic disposal.

1. Sivashanmugam, A., Murray, V., Cui, C., Zhang, Y., Wang, J., & Li, Q. (2009). Practical protocols for production of very high yields of recombinant proteins using Escherichia coli. Protein science, 18(5), 936-948.
2. National Institutes of Health (NIH). NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. National Institutes of Health. Available at: https://osp.od.nih.gov/biotechnology/nih-guidelines/.
3. University of California, Berkeley. Laboratory Safety Manual. University of California, Berkeley. Available at: https://ehs.berkeley.edu/sites/default/files/lab-safety-manual.pdf.
4. National Research Council. Prudent Practices in the Laboratory: Handling and Disposal of Chemicals. National Academies Press, 2011. Available at: https://www.ncbi.nlm.nih.gov/books/NBK55836/.
5. University of Cambridge. Laboratory Safety Guidelines. University of Cambridge. Available at: https://www.safety.cam.ac.uk/guidance/laboratory-safety-guidelines.
6. Centers for Disease Control and Prevention (CDC). Biosafety in Microbiological and Biomedical Laboratories (BMBL) 6th Edition. Centers for Disease Control and Prevention. Available at: https://www.cdc.gov/labs/bmbl/?CDC_AAref_Val=https://www.cdc.gov/labs/BMBL.html