Contribution

During the preparation for our iGEM competition, we not only continuously improved and enhanced our project by designing a novel platform based on CRISPR-Cas9 and DNA origami to treat antibiotic-resistance and inhibit risk of transmission, but also hoped to provide some assistance to future iGEM teams and participants through our efforts. This section introduces the contributions we have made for future teams and members, which involve the scalability of our universal platform FoCas, bio-safety manual and dissemination and educational significance that contributes to society, lists of companies specializing in the production of DNA origami materials, and novel designed procedures for screening aptamers and models.

Scalability of our treatment strategy for infectious AMR

FoCas can act as a universal platform for multiple infectious antimicrobial-resistant bacteria

Antimicrobial-resistant (AMR) bacteria pose a critical threat to public health, especially in nosocomial infections (1). In our project, we designed a universal platform based on CRISPR-Cas9 gene editing system and DNA origami delivery system to specifically eliminate the target resistance gene of the target resistant bacteria. The platform was named FoCas, whose feasibility and functionality have been demonstrated and showcased in our project, using methicillin-resistant staphylococcus aureus (MRSA) as the example target strain. For MRSA, resistance gene mecA was first identified as the target gene through analysis and screening, and sgRNA targeting mecA (sgRNA-ΔmecA) and aptamer targeting MRSA were designed. After initial design, the platform treating MRSA was assembled by incorporating each component: loading component (including designed sgRNA_L-ΔmecA), membrane permeabilization component, targeting component (including designed aptamer), and protection and release component. Then, the complete drug was applied on the wound surface, targeting MRSA, entering the cell, and eliminating mecA gene, resulting in a regained methicillin sensitivity and significantly reduced risk of staphylococcus aureus (Figure 1).

Figure 1. Illustration of simple procedures for FoCas platform

Examples of the platform's strategies on some other candidate high-risk resistant and transmissible bacterial infectious

In many AMR cases, antibiotic resistance genes are evident, and traditional treatments are limited. Examples include methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE), and extended-spectrum β-lactamase–producing Enterobacterales (ESBL-E). Our delivery system can be easily adapted to these diseases, according to the specific genes and membrane proteins involved.

Besides methicillin-resistant Staphylococcus aureus (MRSA), the focus of our current project, other potential targets include:

Vancomycin-Resistant Enterococci (VRE)

Description

Vancomycin-resistant Enterococci (VRE) can lead to blood infections with high morbidity and mortality rates (2). Also, it is inducing increasing nosocomial infections (3). They form opportunistic infections, causing surgical infections through wounds and leading to severe and fatal situations. While treatments such as high-dose daptomycin (≥10 mg/kg) or linezolid often result in better patient outcomes, their long-term efficacy is uncertain.

Gene

There are different types, associated with different "core" genes in different types of bacteria (4). Most common types are VanA and VanB, their regulatory mechanism is well understood. The core gene performs crucial part in the replacement of d‐Ala‐d‐Ala with d‐Ala‐d‐lac, decreasing the binding constant of vancomycin and peptidoglycan, which directly induces high levels of resistance. So, if their "core" genes are removed, this mechanism will cease, and they'll resume sensitivity to drugs.

Parts to be modified

The sgRNA and aptamer need modification according to the specific gene and membrane protein target respectively. Follow our instructions to complete this process.

Extended-spectrum β-lactamase–producing Enterobacterales (ESBL-E)

Description

The rise of antimicrobial resistance from extended-spectrum β-lactamase–producing Enterobacteriaceae (ESBL-E) also poses a significant challenge to global health (5). Extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E) is crucial antimicrobial-resistant organisms for human, and is common reason for urinary tract infections and bloodstream infections (6). Also, it has strong ability to transfer. The ESBL genes are commonly found on transposons or insertion sequences of plasmid that they could spread easily.

Gene

ESBL genes. Though ESBL genes are a large family with different genes, however, as most of them encode enzyme ESBL that leads to drug-resistance, they contain conserved sequences. As long as the crucial and conserved sequences are knocked out, the bacteria may resume sensitivity to the drugs.

Parts to be modified

Same as above.

Despite VRE and ESBL-E, many other drug-resistant bacterial infections also have limited effective treatments. And in cases that pathogenic genes were well-identified our delivery system can be easily modified and utilized.

Bio-safety manual

In vivo drug safety evaluation is essential in drug development, providing crucial insights into a drug's safety and efficacy within a living system, thereby ensuring patient protection and regulatory compliance. We collaborated with Beijing University to establish a manual, providing safety guidelines for drug use in living organisms. This manual can guide future iGEM teams in their work on in vivo drug applications. It could also provide a framework that could impact societal health by advancing personalized medicine and improving drug safety standards.

Dissemination and educational significance

Our team has been to Meitan, Guizhou, for volunteer teaching at Zhejiang university primary school. We taught about DNA and antibiotics in a simple way to stimulate children's curiosity about science. The teaching was successful, with students quickly grasping basic information and gaining a preliminary understanding of infectious diseases and synthetic biology.

Volunteering teaching in Zhejiang university primary school — Figure 2. An image taken during volunteering teaching in Zhejiang university primary school. Our major includes biomedical science, biomedical informatics, electrical and computer engineering, and majors from Zhejiang University International Business School (ZIBS). The background is their main teaching building.

Students from Zhejiang university primary school — Figure 3. Another image taken during volunteering teaching in Zhejiang university primary school. The students from Zhejiang university primary school enthusiastically joined us for photographing.

List of companies specializing in the production of DNA origami materials

Creative Biostructure (America)

Creative Biostructure offers DNA self-assembly services, using DNA origami technology to construct specific nanostructures. The company provides customized DNA origami structure, using advanced design tools such as caDNAno and DAEDALUS for structural optimization and conducts final structural tests through techniques, which is suitable for projects that require highly customized complex DNA nanostructures.

Tilibit Nanosystems (Germany)

Tilibit Nanosystems offers specialized DNA origami structure customization services. Customers can cooperate with the company based on their own needs to develop and optimize DNA sequences to ensure the stability and accuracy of the structure. The company has profound experience in DNA nanotechnology and is suitable for DNA origami projects of all scales and complexities.

Eurofins Genomics (India)

Eurofins Genomics offers customized DNA origami services, including the synthesis and assembly of DNA scaffolds. The company can provide M13mp18 single strand DNA of specific lengths and related DNA origami customization services according to the design requirements of customers. Eurofins Genomics also combines the prefabricated structure library of Tilibit Nanosystems to provide customers with more diverse choices.

Guild BioSciences (America)

Guild BioSciences offers M13mp18 single strand DNA and other DNA origami related services. This company focuses on the field of DNA origami, providing high-quality scaffold DNA suitable for origami experiments of different scales. The company also offers customized services to meet various specific experimental requirements.

Bayou Biolabs (America)

Bayou Biolabs offers the relatively affordable M13mp18 single strand DNA, which is suitable for DNA origami experiments. The company focuses on providing cost-effective DNA origami materials for small laboratories and projects with limited budgets. This company has many global agents, making it very convenient to purchase.

Shaanxi Weishinuo Biotech (China)

Shaanxi Weishinuo Biotech is a company specializing in DNA synthesis and biotechnology services, offering a wide range of DNA scaffold ordering services, including customized M13mp18 single strand DNA.

Procedure for screening aptamers

Our delivery system requires aptamers. After some exploration, we found limited existing procedures for identifying suitable aptamers for our target bacteria. Therefore, we established a standard procedure encompassing molecular dynamics, docking simulation, and artificial selection and evolution.

Models

Our delivery system is nanoscale, but due to computational limitations, highly accurate molecular dynamics simulations may be challenging for our relatively long-duration and high-molecular-weight system. There is also a gap between wet lab experiments and molecular dynamics, as neither can fully achieve the necessary spatial and temporal scales. Our model aims to bridge this gap, benefiting not only our research but also the entire iGEM community. You may find inspiration for preliminary analysis and prediction in your own nanoscale designs. For example, our model simulating membrane penetration could be applied to other experiments involving "membrane damage."

Additionally, describing the protective effect of carriers on payloads at the nanoscale can be challenging. Our model addresses this from the perspective of chemistry and physics, and we introduced physical formulas and set coefficients to represent chemical influences.

References

van Duin D, Paterson DL. Multidrug Resistant Bacteria in the Community: An Update. Infect Dis Clin North Am. 2020 Dec;34(4):709–22.
Cairns KA, Udy AA, Peel TN, Abbott IJ, Dooley MJ, Peleg AY. Therapeutics for Vancomycin-Resistant Enterococcal Bloodstream Infections. Clin Microbiol Rev. 36(2):e00059-22.
Mareković I, Markanović M, Lešin J, Ćorić M. Vancomycin-Resistant Enterococci: Current Understandings of Resistance in Relation to Transmission and Preventive Strategies. Pathogens. 2024 Nov 5;13(11):966.
Stogios PJ, Savchenko A. Molecular mechanisms of vancomycin resistance. Protein Sci. 2020 Mar;29(3):654–69.
Husna A, Rahman MM, Badruzzaman ATM, Sikder MH, Islam MR, Rahman MT, et al. Extended-Spectrum β-Lactamases (ESBL): Challenges and Opportunities. Biomedicines. 2023 Oct 30;11(11):2937.
Peirano G, Pitout JDD. Extended-Spectrum β-Lactamase-Producing Enterobacteriaceae: Update on Molecular Epidemiology and Treatment Options. Drugs. 2019 Sept 1;79(14):1529–41.