<strong>Safe handling of cell lines:</strong> The cell lines used for experiments were handled in accordance with the applicable safety regulations. This included regular checks for contamination and the safe storage and disposal of cell cultures.
</p>
<H4text="Checkin for the Prime-Editing Komplex "></H4>
<H4text="Check-in for the Prime-Editing Komplex "></H4>
<p>
<strong>Reverse transcriptase:</strong> Reverse transcriptase plays a central role in prime editing by specifically inserting the correction as DNA at the inserted nick using an RNA template provided by pegRNA. The correction of the complementary DNA strand then takes place via the natural cell repair mechanisms. This ensures an exact correction of the target sequence. We checked the reverse transcriptase to ensure it could perform precise genome editing without introducing unintended mutations. This was important to minimize the risk of off-target effects that could lead to unexpected or harmful consequences.
This prime-editing complex thus represents a precise and efficient method for gene editing. By combining these components, genetic modifications can be performed with minimal side effects
</p>
<H4text="Checkin for Cloning"></H4>
<H4text="Check-in for Cloning"></H4>
<p>
For our cloning experiments and the development of our prime editing complexes, we have amplified various plasmids in <i>E. coli</i> K-12 strains (DH5α,10-Beta) When working with microbial strains such as <i>E. coli</i> K-12 strains, a it's important to consider potential risks associated with their use, even though they are generally regarded as safe in laboratory settings. All experiments were performed under strict S1 conditions, following all relevant safety protocols. Below you will find an overview of the <i>E. coli</i> K-12 strains for our cloning experiments, submitted by us as a checkin and the specific safety measures:
<strong><i>Pichia pastoris</i> (SMD1163):</strong><i>Pichia pastoris</i> (SMD1163) is a widely used yeast strain for the expression of recombinant proteins. It is characterized by a methanol-inducible expression system (AOX1 promoter) and high cell growth rates, which makes it ideal for industrial applications. The strain can be easily genetically manipulated and can perform post-translational modifications, which supports correct protein production.
When working with <i>Pichia pastoris</i> (SMD1163), various safety-relevant aspects must be observed. Although the organism is considered non-pathogenic and biologically safe (S1), skin contact and aerosol formation should be avoided to minimize the risk of infection or allergic reactions. When using genetically modified strains, it is important to follow the relevant GMO guidelines to prevent uncontrolled release. In addition, handling chemicals such as methanol requires special precautions as they are toxic and highly flammable. The disposal of cell cultures and waste must also be carried out in accordance with biosafety regulations, especially in the case of genetically modified organisms.
</p>
<H4text="Checkin for Testing in cell lines "></H4>
<H4text="Check-in for Testing in cell lines "></H4>
<p>
In our project, we paid attention to safety at every step, especially when working with specific cell lines [link Zellinien]. All experiments were performed under strict S1 conditions, following all relevant safety protocols. Given the sensitivity of the human cell lines we used, we placed great emphasis on controlled and well-designed workflows. All transfections were performed in our own transfection laboratory to ensure a high level of safety and compliance. Below you will find an overview of the cell lines submitted by us as a checkin and the specific safety measures:
As part of our laboratory activities for our PreCyse project, we worked in various laboratories. For general lab work and cloning experiments, you can find some pictures of our laboratories below:
</p>
<H4text="Our Cloning Lab"></H4>
<p>
Our Cloning-laboratory is divided into different work areas to ensure that the experiments run smoothly and efficiently. These include the gel station, the PCR station, the transformation section and the measurement area. Each area is specially equipped for the respective method, and the corresponding experiments were carried out exclusively in the designated stations. In this way, we ensure that our work is carried out under optimal conditions and with the greatest possible precision.
</p>
<H4text="Our Cell Culture Lab "></H4>
<p>
In our cell culture laboratory, we work under sterile conditions to ensure optimal growth conditions for human cell lines. Among other things, we carry out transfections in order to introduce genetic material into cells and investigate their behavior. Strict protocols and state-of-the-art technology ensure the precision and reproducibility of our experiments.
</p>
<p>
In our S2 laboratory, the harvested nasal epithelial cells that serve as primary cultures undergo a comprehensive HHH test (link zu primär Kulturen) to ensure their safety and suitability for further experiments. This test is crucial to ensure that we can subsequently work safely with these cells in the S1 range without the risk of contamination or unwanted release of biological material.
Our project focuses on the genetic disease cystic fibrosis, specifically targeting the Delta-508 mutation. The aim is to correct this mutation using Prime Editing, a precise genome-editing technique. We have explored different strategies to optimize the Prime Editing complex for this specific application.
</p>
<p>
The Prime Editing complex consists of a nickase, a reverse transcriptase, a pegRNA.. The pegRNA guides the editing process by directing the complex to the target DNA sequence, allowing for precise genetic modifications. For targeted delivery, we selected lipid nanoparticles (LNPs) to introduce the mRNA encoding the Prime Editing components specifically into lung epithelial cells, where the CFTR protein is highly expressed. Additionally, we investigated alternatives to the conventional Cas9 nickase, such as the smaller CasX and Fanzor, aiming to reduce the overall size of the Prime Editing complex. In our optimization efforts, we also explored smaller reverse transcriptases to enhance the efficiency of the system in human cells.
The Prime Editing complex consists of a nickase, a reverse transcriptase, a pegRNA. The pegRNA guides the editing process by directing the complex to the target DNA sequence, allowing for precise genetic modifications. For targeted delivery, we selected lipid nanoparticles (LNPs) to introduce the mRNA encoding the Prime Editing components specifically into lung epithelial cells, where the CFTR protein is highly expressed. Additionally, we investigated alternatives to the conventional Cas9 nickase, such as the smaller CasX and Fanzor, aiming to reduce the overall size of the Prime Editing complex. In our optimization efforts, we also explored smaller reverse transcriptases to enhance the efficiency of the system in human cells.
</p>
<p>
Furthermore, we have developed a modular plasmid that contains the backbone of our Prime Editing complex. The individual components can be cloned individually into the backbone. This plasmid allows us to either deliver the construct directly into target cells or transcribe the plasmid into RNA, enabling the delivery of the Prime Editing complex in the form of mRNA. The modularity of the plasmid is a key feature; specific restriction sites are included to facilitate the easy exchange of the complex's components. This design makes it straightforward to adapt the Prime Editing complex for various use cases and therapeutic requirements.
To mitigate the risk of dual-use, where our Prime Editing technology could be exploited for unintended and potentially harmful purposes, we have implemented several strategies.
Together, these measures provide a layer of protection against dual-use risks, making it more difficult for the technology to be employed outside of its intended therapeutic context.
</p>
<p>
<strong>Unitended Dissemination</strong>
<H5text="Unitended Dissemination"></H5>
</p>
<p>
To minimize the risk of our construct being inadvertently released into the environment, we adhere to strict standard operating procedures (SOPs) in the laboratory. These SOPs are designed to ensure that all safety standards are maintained, particularly when handling LNPs) and genetic materials. This includes rigorous protocols for storage, disposal, and decontamination, reducing the chance of accidental dissemination of the materials we work with.
Together, these practices and design strategies help to ensure that the risk of our constructs inadvertently affecting non-target organisms or environments is minimized.
</p>
<p>
<strong>Unauthorized Access</strong>
<H5text="Unauthorized Access"></H5>
</p>
<p>
To prevent unauthorized access to our genetic sequences, plasmid constructs, and other sensitive information, we have implemented strict internal biosecurity policies. These policies ensure that we carefully manage which materials exist in physical form and their exact storage locations.
Furthermore, we ensure that any sensitive data generated during our project is only published in the supplementary materials section, which is accessible exclusively to those with an official iGEM account. By taking this approach, we can limit the availability of sensitive information and reduce the risk of it being misused.
</p>
<p>
<strong>Synthetic Biology and Information Sharing</strong>
<H5text="Synthetic Biology and Information Sharing"></H5>
</p>
<p>
Managing the risks associated with synthetic biology and information sharing is a complex challenge. Many of the strategies we use to address other risks, such as "Unauthorized Access" and "Dual-Use Potential," are equally relevant in this context. For instance, our strict control over access to sensitive information and our safety mechanisms both help mitigate the risks posed by the accessibility of synthetic biology tools.
To address this, we adhere strictly to all policies and regulations set forth by iGEM, our university, the German government, and the European Union. By aligning our practices with existing regulations on synthetic biology, we aim to conduct our work responsibly and reduce the risks associated with the field.
</p>
<p>
<strong>Public Perception and Miscommunication</strong>
<H5text="Public Perception and Miscommunication"></H5>
</p>
<p>
One of the challenges in scientific research, especially in fields like synthetic biology, is that advancements can often outpace public understanding and discourse. This can lead to confusion, fear, or mistrust if the research is not communicated effectively. To address this issue, we believe that scientific progress should occur in constant dialogue with the public.
