<H4id="edu-heading"text="Our education and outreach"/>
<H4id="edu-heading"text="Our education and outreach"/>
<LoremMedium/>
<p>Education and outreach remain vital, even without being a special prize focus. They help us share knowledge, inspire people, and create lasting impact. By making information accessible, we empower the public to make informed decisions and engage in meaningful discussions.</p>
<p></p>
<H4id="edu-why-heading"text="If not as a special prize - then why?"/>
<H4id="edu-why-heading"text="If not as a special prize - then why?"/>
<LoremMedium/>
<p>Human-centered design is essential for our Integrated Human Practices, ensuring that our project is aligned with the real needs and concerns of the people it aims to serve. Outreach and education are key components of this approach, as they allow us to communicate our goals and inform the public about both our project and the critical issue of cystic fibrosis (CF). By engaging with diverse audiences—patients, families, schools, and the wider community—we raise awareness about CF, while fostering a deeper understanding of the challenges involved. This feedback-driven interaction not only builds trust but also helps us refine our work to have a meaningful impact on those affected by CF. Through educational programs, workshops, and outreach initiatives, we aim to bridge the gap between scientific research and the public, ensuring that our project is both accessible and impactful.</p>
<H4id="student-academy-heading"text="Student academy on the topic of synthetic biology"/>
<H4id="student-academy-heading"text="Student academy on the topic of synthetic biology"/>
<H5id="Schüler*innen Akademie"text="Teaching the Next Generation of SynBio Pioneers "/>
<H5id="Schüler*innen Akademie"text="Teaching the Next Generation of SynBio Pioneers "/>
<p> The Center for Biotechnology (CeBiTec) at Bielefeld University organizes the annual CeBiTec Student Academy for “Biotechnology and Biomedicine.” Supported by the Osthushenrich Foundation and the Detmold district government, the academy offers students a unique opportunity to deepen their understanding of biology, genetics, and molecular biology through hands-on experiments and expert lectures. Key topics include nanopore sequencing, tumor diagnostics, and the evolution of SARS-CoV-2. The program is especially valuable for students transitioning from school to potential studies in the natural sciences.
<p> The Center for Biotechnology (CeBiTec) at Bielefeld University organizes the annual CeBiTec Student Academy for “Biotechnology and Biomedicine.” Supported by the Osthushenrich Foundation and the Detmold district government, the academy offers students a unique opportunity to deepen their understanding of biology, genetics, and molecular biology through hands-on experiments and expert lectures. Key topics include nanopore sequencing, tumor diagnostics, and the evolution of SARS-CoV-2. The program is especially valuable for students transitioning from school to potential studies in the natural sciences.
Due to our collaboration with the Student Academy, we conducted the nanopore sequencing experiment and served as teachers, assisting in experiment preparation, execution, offering guidance, and answering questions. This role allowed us to teach the students about laboratory work, the critical aspects of conducting experiments, and essential safety considerations. The experiment involved isolating bacterial DNA, preparing samples for sequencing, and performing both sequencing and data analysis.</p>
Due to our collaboration with the Student Academy, we conducted the nanopore sequencing experiment and served as teachers, assisting in experiment preparation, execution, offering guidance, and answering questions. This role allowed us to teach the students about laboratory work, the critical aspects of conducting experiments, and essential safety considerations. The experiment involved isolating bacterial DNA, preparing samples for sequencing, and performing both sequencing and data analysis.</p>
<p>Since we presented our iGEM project PreCyse to them as well, the students were introduced to study-related projects like iGEM. They learned about the daily tasks, challenges, and responsibilities involved in iGEM through project discussions. Many students were captivated by the iGEM concept and expressed interest in participating during their future studies. They were particularly fascinated by the opportunity to develop real research projects, work independently in the lab, learn extensively about synthetic biology, and implement creative ideas while collaborating with an international team.</p>
<p>Since we presented our iGEM project PreCyse to them as well, the students were introduced to study-related projects like iGEM. They learned about the daily tasks, challenges, and responsibilities involved in iGEM through project discussions. Many students were captivated by the iGEM concept and expressed interest in participating during their future studies. They were particularly fascinated by the opportunity to develop real research projects, work independently in the lab, learn extensively about synthetic biology, and implement creative ideas while collaborating with an international team.</p>
aimofcontact:"Shortly after we decided to use prime editing as the gene editing method for our cystic fibrosis therapy, Mattijs Bulcaen from the Laboratory of Molecular Virology and Gene Therapy at KU Leuven and his colleagues published a paper directly related to our research[1]. In contrast to our approach, Bulcaen et al. 2024 targeted other, less common but drug-refractory CFTR-specific mutations (L227R- and N1303K). ",
aimofcontact:"Shortly after we decided to use prime editing as the gene editing method for our cystic fibrosis therapy, Mattijs Bulcaen from the Laboratory of Molecular Virology and Gene Therapy at KU Leuven and his colleagues published a paper directly related to our research[1]. In contrast to our approach, Bulcaen et al. 2024 targeted other, less common but drug-refractory CFTR-specific mutations (L227R- and N1303K). ",
insights:"The interview with Mattijs was valuable for us in a lot of ways. At that point in the project we were starting to design the components of our prime editor, but we were lacking a broader overview over the state of the field. Mattijs gave us this insight, mentioning techniques like PE3b systems, dsgRNAs and a talk given by David Liu,[Link] the principal investigator behind prime editing that helped us to consider further novel advancements in in Prime Editing and include them into our project. He discussed with us the difficulties that might await us when targeting the CFTR F508del deletion and mentioned that insertions of all the edits possible with prime editing are the hardest to make, the recognition of edits in the region might attract mismatch repair systems and the chromatin organization might negatively impact prime editing efficiency. Also, we learned a lot about how to design our pegRNAs, with important inputs being the 3’ stem loop motif trevopreQ1 used by Mattijs in his publication and the suggestion to use prediction tools to evaluate sgRNA spacer cutting efficiency. We reviewed our approach of testing pegRNAs using the PEAR reporter system and Mattjis recommended to use HEK cell lines for screening because of their easy handling and naturally impaired mismatch repair system. ",
insights:"The interview with Mattijs was valuable for us in a lot of ways. At that point in the project we were starting to design the components of our prime editor, but we were lacking a broader overview over the state of the field. Mattijs gave us this insight, mentioning techniques like PE3b systems, dsgRNAs and a talk given by David Liu,[Link] the principal investigator behind prime editing that helped us to consider further novel advancements in in Prime Editing and include them into our project. He discussed with us the difficulties that might await us when targeting the CFTR F508del deletion and mentioned that insertions of all the edits possible with prime editing are the hardest to make, the recognition of edits in the region might attract mismatch repair systems and the chromatin organization might negatively impact prime editing efficiency. Also, we learned a lot about how to design our pegRNAs, with important inputs being the 3’ stem loop motif trevopreQ1 used by Mattijs in his publication and the suggestion to use prediction tools to evaluate sgRNA spacer cutting efficiency. We reviewed our approach of testing pegRNAs using the PEAR reporter system and Mattjis recommended to use HEK cell lines for screening because of their easy handling and naturally impaired mismatch repair system. ",
implementation:"The inputs given by Mattijs directly impacted our design choices for multiple parts of the project. For the pegRNA design, we decided to use the same 3’ motif as Mattijs had used and also, like he suggested, checked our spacer candidates for predicted cleavage efficiency. Also we used HEK cells for screening our pegRNAs. We looked further into PE systems that influence cellular mismatch repair (such as PE4) and tried to include these into our design. ",
implementation:"The inputs given by Mattijs directly impacted our design choices for multiple parts of the project. For the pegRNA design, we decided to use the same 3’ motif as Mattijs had used and also, like he suggested, checked our spacer candidates for predicted cleavage efficiency. Also we used HEK cells for screening our pegRNAs. We looked further into PE systems that influence cellular mismatch repair (such as PE4) and tried to include these into our design. ",
interview:<>
<QaBoxq="You mentioned that it was quite challenging to target the F508 delta mutation. Could you provide more detailed reasons for why this is the case or explain why this mutation is particularly difficult to target compared to others?"a="Yes, that's the million-dollar question. First of all, let me clarify: our group has never directly worked on that mutation because we immediately focused on the drug-refractory mutations, such as nonsense mutations where the protein is not formed, indel mutations, or severe missense mutations that do not respond to modulator therapies. Of course, we know several groups in the field who either work on gene editing or focus on cystic fibrosis (CF). We've heard from some of them who attempted to target the F508 delta mutation. For example, some collaborators really tried to design different guides but were unable to find anything above the detection limit. F508del is probably one of the most logical mutations to try to correct, not just for CF but for the entire gene-editing field. If you look at the frequencies of mutations that cause genetic diseases, the F508 delta mutation is by far the most common deletion mutation causing a severe disease. This is because CF, along with sickle cell disease, is one of the most common deadly inherited diseases, and it's overrepresented within CF. So, it makes sense that they would have been trying to target it from the beginning. Interestingly, if you read the Prime Editing paper by Anzalone, F508 delta is mentioned in the introduction in connection with cystic fibrosis. So, it's somewhat surprising that after all this time—it's been almost five years now—they haven't published or released anything on F508 delta. However, last weekend, there was an online seminar where David Liu gave a talk, and he showed some unpublished data indicating that they managed to achieve quite good Prime Editing efficiency on F508 delta. It's worth noting that David Liu rarely presents unpublished data unless the publication is either accepted or very close to acceptance. So, we all kind of expect that the paper will be published soon, perhaps within the next week or at least within a month. From what I saw, it appears they leveraged many of the approaches available today to enhance Prime Editing. Now, regarding your question about why this mutation is so difficult to target with Prime Editing, I can't provide an exact answer. However, I can list some potential difficulties associated with the mutation, and it’s likely that F508 delta is challenging for several of these reasons. For instance, it could be related to the genomic region itself. Writing insertions can be more difficult; the easiest edits are single-point mutations, followed by deletions, and the most challenging are insertions. This difficulty arises because it involves writing a third strand and then relying on DNA damage repair mechanisms to fix it. It could also be that the region around the F508 delta mutation is challenging due to flap equilibration or that it attracts pathways such as mismatch repair that negatively impact Prime Editing. Additionally, the chromatin organization around that region could play a role. Over the past year, we’ve gathered clues that chromatin organization significantly affects Prime Editing capability, while this is much less of an issue for Cas9 and base editors. Studying this is not straightforward; you would need to conduct experiments like ATAC-seq to determine the chromatin organization around the mutation and how it might interfere. I also noticed on a slide that dsgRNAs were mentioned, though David Liu didn't discuss them in his talk. After looking them up online, I found that this technique, published a few years ago by other researchers, is specifically designed to open up chromatin. It seems they use different guides, without the three-prime extension, to open up the chromatin, which could be one way to overcome the limitations in Prime Editing efficiency. There could be other factors as well, and it’s often difficult to predict what will work and what won't. We have prediction tools for Prime Editing guides that work to some extent, but they are not as effective as the prediction tools available for regular CRISPR guide RNAs. This suggests that the Prime Editing system is more complex than the canonical CRISPR systems, with more variables that can influence success or failure. I hope this answers your question somewhat."/>
<QaBoxq="Perhaps we could quickly discuss which part of the prime editing complex you think plays the most significant role in making insertions much more challenging compared to deletions. Is it the reverse transcriptase or the RNA?"a="I don't think it's primarily the reverse transcriptase that's the issue. People have shown that longer insertions are definitely possible. I believe the challenge lies in the process when your cell has to repair the new DNA strand, which is generated and exists as a three-stranded intermediate. We don’t directly intervene in this process; it entirely depends on the cell and the DNA damage repair pathways active in those cells. Through expression of dominant negative DNA damage repair effectors, or by nicking the non-edited strand, the outcome can be steered to some extent. When you perform an insertion, the new strand must hybridize with the bottom strand, which remains intact. This creates a small loop that needs to be incorporated. At this point, the cell faces two options: it can either revert to the original state or incorporate the edit you’re trying to introduce. In certain circumstances, perhaps due to how the new DNA strand folds or the sequence context of the region of interest, the cell might heavily favor reverting to the original state, resulting in the absence of the intended edit. This process is extremely difficult to predict, but there are several indications pointing in this direction. For example, in the case of point mutations, it has been shown that it’s easier to convert a C to a G rather than the reverse, simply due to how these mismatches are recognized by the DNA damage repair mechanisms. This area is very complex, and I don’t think anyone fully understands it yet. It’s also difficult to study. I don't believe the rate of reverse transcription is the limiting factor here, although it could play a role for long or structured pegRNAs. You might have already come across this, but the PE6 generation of Prime Editors, which were released about half a year ago, involve engineered or evolved reverse transcriptases that are more processive and can more easily synthesize longer transcripts. Another factor that could play a role is the secondary structure of the guide RNA. Each prime editing guide RNA faces a common problem: it has a spacer that binds the bottom strand and a three-prime extension that binds the top strand. Since these two parts of the RNA bind complementary strands, they are also complementary to each other, meaning every prime editing guide has some tendency to bind itself. If the Gibbs free energy is too high, the guide RNA may fold in on itself, preventing it from binding to the prime editor, which then inhibits prime editing. Additionally, the three-prime extension itself can fold independently. I haven’t specifically examined this for the F508 delta guides, but it is something that can be predicted. There are tools available that can predict the secondary structure of an RNA sequence, and if there’s a significant hairpin structure, it might mean the three-prime extension remains closed, preventing the reverse transcriptase from using it as a template. The PE6 prime editors have been engineered to be more effective in such scenarios, being less affected by secondary structures and better able to read through them."/>
<QaBoxq="What would be the application? Would you administer the heat shock in vivo, or...?"a="I believe they used it to engineer zebrafish embryos or something along those lines. It’s quite specific, of course. If you plan to deliver your guide RNA through a viral vector or similar method for human therapy, the application would differ significantly. You obviously can't administer a heat shock to humans, so it really depends on the context of your application."/>
<QaBoxq="Given the time constraints, let's move on to the next question. Due to our limited resources, we are targeting a PE2 system, and we'd like to ask if you see any chances of success with this system. If so, how high do you think the chances of success are?"a="PE2 can work, but it really depends on your application and the methods you have to assess the editing efficiency. If you can use NGS (Next-Generation Sequencing) for everything, you'll be able to detect edits even with PE2 systems. However, I would generally expect the efficiency to be low. Whenever possible, I would always recommend trying the PE3 system. Could you share what your specific application is, or is that confidential?"/>
<QaBoxq="So our goal is to eventually use it in vivo, but for now, we're focusing on trying to correct the mutation first in regular cell cultures and then later in primary cells."a="Is your focus specifically on the F508 delta mutation? If so, we could potentially help you get you started, as we already have constructs and cells with that mutation. We would need to discuss the financial aspects, but we might be able to assist. However, are you fully committed to targeting F508, or are you also considering other diseases or mutations?"/>
<QaBoxq="The timeframe of the project, combined with the fact that we’re all studying on the side, limits us to a certain scope. Since this is our first time tackling a project like this, it makes sense to stick to something more manageable. So, we're somewhat committed to focusing on F508 due to these constraints."a="That's understandable. It can be really tough to juggle a project like this along with exams and studies, especially if you're also involved in competitions. But it's definitely worth the effort, even if you don't achieve huge results right away. The experience and learning, as well as the connections you make, are incredibly valuable. I'm a big supporter of such"/>
<QaBoxq="We have one patient who is willing to provide us with cells, but we don't have them yet."a="It sounds like you're aware of the challenges, and I don't want to discourage you, but just to be realistic, working with primary cells and getting everything ready could be tricky, especially considering the competition is in October. Experiments in human cells can take time, especially if you need to do multiple iterations or clone constructs—it could easily take a week or more per experiment."/>
<QaBoxq="Regarding the cells we have, as mentioned in our paper, we screened all our guides on HEK cells with an integrated copy of the CFTR cDNA. HEK cells are easy to work with, but they don't naturally express CFTR, even though the gene is present in their genome. So, we introduced the mutation of interest into these cells, making it easier to screen."a="I'm not entirely sure if we can send over the cells due to ethical regulations, which can be complex and time-consuming to navigate. However, there's an alternative approach that might help you. Early on, we found that it's actually quite easy to screen guides using what we call a 'transient target.' In this method, you would transfect all your prime editing plasmids into HEK cells, along with a plasmid containing the CFTR cDNA with the mutation of interest."/>
<QaBoxq="While this approach isn’t as physiological as editing the chromosome directly, our side-by-side comparisons showed almost equal efficiencies between transient and chromosomal targets. It's much easier and faster than working with patient-derived cells. I can definitely send you the plasmid, which would save you a lot of time and effort. This method is much simpler and could be a practical solution for your project."a="Our initial plan is to work with a reporter plasmid that expresses eGFP, where we've removed a splice site, until we have patient cells or cell lines with CFTR mutations. This will allow us to screen easily without needing to sequence everything. Do you maybe have any suggestions or advice on this approach?"/>
<QaBoxq="Is that the PEAR system?"a="No, it’s a different one, but we also have a similar system. The advantage of this approach is that you can very easily see if it works, and it’s very sensitive—much easier than extracting and sequencing DNA. The downside, however, is that… actually, I’m not familiar with the 'flu PEAR system.'"/>
<QaBoxq="Actually, we use the exact same system in our lab. It’s very useful for optimizing delivery strategies because it’s easy to see results. The downside, of course, is that the guides you’re using for that system aren’t specific to the F508 delta mutation, right? So, these are scientific trade-offs. You could, for example, design a reporter that uses your F508 delta guide and also results in fluorescence, but you would need to design the reporter first. It’s challenging to prove that it works because you might not have a perfect guide for F508 delta."a="It really depends on what you want to achieve. If your goal is to first check if you can successfully perform prime editing, then using the reporter is definitely a good first step."/>
<QaBoxq="We will edit the plasmid, specifically the vector, so that we have almost the same pegRNA. The only difference will be downstream, behind the edit."a="Is this approach based on a paper from the Netherlands, or is it something you came up with yourself?"/>
<QaBoxq="Based on a paper."a="Yeah, that sounds like a very good way to start. Do you already have the reporter plasmid ready?"/>
<QaBoxq="Yeah, we bought the reporter, and now we’re making the necessary edits so we can use it."a="Okay, so do you also already have guides targeting F508 right now?"/>
<QaBoxq="We’ve designed some guides, but we haven’t tested them yet. That’s one of our next steps. So, at the moment, we’re just in the design phase, or we have already designed them, and..."a="Yeah, okay, cool. Good luck with that! And I suppose you’re starting off with HEK cells as well, right?"/>
<QaBoxq="We have HEK and HeLa cells, but we haven't decided yet which ones we'll use."a="I would start off in HEK cells because, by total accident or coincidence, they are much easier to achieve prime editing in. This is because the MLH1 gene, which negatively impacts prime editing outcomes, is naturally disabled in these cells—they don't produce the MLH1 protein. Of all cell lines available, HEK cells are the easiest to achieve editing with, so I would definitely recommend starting there."/>
<QaBoxq="In terms of transfection, HEK cells are also very easily transfected. If I can offer another piece of advice, always include GFP controls—plasmids that simply express GFP without requiring editing—and use them to determine your transfection efficiency. It's crucial to have a very high transfection efficiency because you'll be working with a three-component system: your reporter, your prime editor, and your guides. All three plasmids need to be present in the same cell for the editing to occur, so you should aim for at least 70% transfection efficiency, preferably 80% or higher."a="I don't know what transfection method you're planning to use, but we've always used Lipofectamine 3000. It’s expensive, but it works very well. However, if you're looking for more cost-effective options, we recently discovered two other transfection reagents, Jet Optimus and Jet Prime, which are much cheaper and also work quite well. That said, I would advise against starting with any of the cheaper transfection reagents; you really need to aim for high transfection efficiency."/>
<QaBoxq="Always make sure to measure and report transfection efficiency for every experiment because if it's low, the experiment might not yield useful results. If you have the funds or resources, I would also recommend designing P3 or even P3b guides, as they might offer better efficiency."a="When it comes to designing P3b guides, if you're primarily focused on P2 right now, there are some specific considerations to keep in mind. I'll provide you with a site that can help with this, and I'll give you the link in just a moment."/>
<QaBoxq="So, it's very advisable to check the Doench score. Do you know what it is?"a="No, not really."/>
<QaBoxq="There are papers by John Doench, an American researcher, from quite a while ago that, in my opinion, are some of the best around. He developed a comprehensive scoring matrix specifically for regular Cas9 that can evaluate the quality of the spacer in your guide RNA. This is important because Cas9 tends to prefer certain sequences over others. For instance, a good spacer should have an appropriate GC content and should avoid hairpins that might cause it to fold in on itself, which would prevent it from functioning properly. You can use this matrix to give a score for the quality of a guide RNA. I’m going to pull up an example here. The site from Synthego, a commercial provider of CRISPR reagents, allows you to check the quality of your guide. When you validate it, the site gives a score based on various factors, including off-target effects, although that might not be your primary concern at the moment. If you hover over a specific area, it will show you the Doench Score, which is crucial. Ideally, you want a guide with a good Doench Score. A good score starts at around 0.4, indicated by a green check mark for good efficiency. If the score is very low, it means that the guide likely has low CRISPR-Cas9 activity and may not be very efficient. When designing prime editing guides, RNA, we always check the spacer for a good Doench Score. If we are designing nicking guides for a PE3 or PE3b strategy, we also ensure that they have a good score. This is one of the easiest tools to check for that. Whenever possible, try using PE3. In some cases, PE3 performs better than PE2, though not always. PE3b might not always work either, but for many mutations, we have seen significant increases in editing efficiency by including the PE3 guide."a="Okay, yeah, that was quite clear from your results; the diagram illustrated that very well."/>
<QaBoxq="Are there more off-target effects when using PE3 since you have to make another cut?"a="If you decide to use PE3, it's important to be aware that while it's not exactly an off-target issue, there is a risk of an undesired on-target outcome. The concern with regular PE3 is that both strands of DNA can be nicked simultaneously, which can lead to a staggered double-strand break. This can result in the formation of indels (insertions or deletions). In your case, this means that if the region around the F508 delta mutation is broken, the prime editor might not be able to repair it properly, leading to additional base pairs being removed or added, and thus, the sequence might be altered in an unintended way. The risk of on-target indels is definitely higher with PE3 compared to PE2. However, this risk is reduced when using PE3b, which employs sequential nicking. The PE3b nicking guides are designed to recognize the wild-type sequence, and they can only nick the opposite strand if the correction has already been made on the top strand. This sequential action helps to avoid the generation of indels. Introducing a second guide into the system also brings the possibility of off-target editing by that guide however, since only a Cas9 nickase is used, off-target indels should be limited."/>
<QaBoxq="Yes, okay, thank you. Do you have time left, or are we out of time?"a="It's fine."/>
<QaBoxq="We have more or less one last question. If it’s not possible, that’s completely fine. We just wanted to ask if you could possibly forward the contact details for the Ussing chamber setup in Paris that you mentioned in your email. Would that be possible?"a="You can certainly try to contact them, but I actually know that there are quite good labs in Germany that work on similar things."/>
<QaBoxq="One major drawback for you might be the time it takes to differentiate cells. If you harvest stem cells or basal cells from patients, they will have the CFTR gene, but they don’t express it immediately. It takes about four weeks for them to differentiate and start producing the CFTR protein. Without this differentiation, you can't measure the currents, which could slow you down significantly. I'm not sure if you have that kind of time."a="If I can give you one piece of advice: it’s less physiological, but it’s still an accepted assay—try it on organoids. We could actually perform both assays here. If you find guides that work really well, we could consider doing those tests here. Someone could come over, or we could do the experiments if they’re not too expensive and have a good chance of working. I think we wouldn’t mind adding the F508 delta mutation to our list of editable mutations."/>
<QaBoxq="There’s also the possibility that if the paper from the Liu Lab is published within the next month, you could just use the guide they provide, and you’d have a guide that is known to work."a="Yeah, so I think if our guides don’t work as well as we hope, this could be an opportunity. We still want to explore optimization of the prime editing system, such as trying different reverse transcriptases or other methods. For now, we’d like to try it on our own, but like you said, it’s good to have this opportunity in case it doesn’t work out."/>
<QaBoxq="Yeah, I think working with patient cells is one thing, but just be aware that these models and assays typically take a lot of time—easily half a year, and that’s considered fast to get them up and running. Unless you're in a lab that already has experience with growing organoids, it could be very challenging to start from scratch."a="However, you can always try. The team in Paris that we know very well—they are incredibly kind, world-class experts in what they do, but they are also under a lot of pressure. They use these technologies not only for research but also to diagnose patients. What the French team has managed to do is show that if a patient’s cells respond to certain drugs, the government allows those drugs to be administered to the patient. You can imagine how important these experiments are, as they can directly impact patients' lives, which naturally takes the highest priority."/>
<QaBoxq="Yeah, we recognized that too. We talked with the CF team at the University Clinic in Münster and asked about using their Ussing chamber, but they are really overworked with it. That’s why we reached out to you about it. But it’s completely fine, as we mentioned before."a="I'm going to put it bluntly: Ussing chamber experiments, while they are highly regarded and provide valuable data, are a real pain to perform. They are incredibly time-consuming and have a very low throughput. A typical setup has four chambers, so you always need to do repeats. In the best-case scenario, you can test two conditions at a time. If you have a very experienced person, they might be able to run eight samples, but they would have to stay with the machine for four to five hours, maintaining constant attention. With multiple technicians, as is the case in France, you might manage to run 16 samples a day."/>
<QaBoxq="On top of that, the cells need to be differentiated properly, and you have to know how to handle them correctly. The medium required is very expensive, and working with these cells is almost more of an art than a science. You have to know when the cells look 'happy' or not because you don't want to waste time on cells that aren't in good condition. I've run quite a few of these assays myself, and while they are great for CF work and provide results that are relevant to patient outcomes, they are technically challenging and very demanding."a="If you want a functional output to show that the CFTR protein is working again, I would recommend starting with one of the easier models, like organoids. We also have in our lab 16HBE cells with a YFP sensor. I don't know if you've heard or read about that. These cells express YFP, which is sensitive to halide ions, including chloride and iodide. When you add a buffer containing these ions to the cells, the YFP intensity quenches. This is something we typically use in our experiments."/>
<QaBoxq="For wild-type cells, you see a rapid and dramatic quenching because CFTR allows these ions to enter the cells. In cells with the mutation, there’s no quenching because the channel isn’t working. While it’s less relevant because these aren't patient cells, it’s closer to reality. The 16HBE cell line is an airway epithelial line, and the expression of CFTR is endogenous, so it’s not at the exaggerated levels you might see in more artificial models like HEK cells."a="Using the YFP assay could be a good alternative or a Plan B for getting a functional readout. This assay is medium to high throughput—you can run entire 96-well plates in about half an hour. All you need for this is the cells and a plate reader that can measure fluorescence and inject the buffer. If you don’t have a plate reader with an injection system, you can also manually add the buffer and quickly place the plate in the machine."/>
<QaBoxq="Yes, that sounds quite good. I think we’ll definitely consider that as a method."a="If you have a little more time, I wanted to ask about the pegRNA. You stabilized it with a stem loop or some kind of motif in the paper, like the trevopreQ1. Did you test other motifs as well, or...?"/>
aimofcontact:"We contacted the organization CF vests worldwide [Link] with the aim to hear more diverse perspectives beyond Germany. After the founder Rod connected us with Joshua, Joshua was so kind to conduct an interview with us not only about the perspectives and stories he heard but also about his personal experiences with his daughter and living in a country where CF care is very hard to get. Joshua (from the USA) and his family live in Thailand where he and his wife run a children’s home. Their daughter is the only child with CF.",
aimofcontact:"We contacted the organization CF vests worldwide [Link] with the aim to hear more diverse perspectives beyond Germany. After the founder Rod connected us with Joshua, Joshua was so kind to conduct an interview with us not only about the perspectives and stories he heard but also about his personal experiences with his daughter and living in a country where CF care is very hard to get. Joshua (from the USA) and his family live in Thailand where he and his wife run a children’s home. Their daughter is the only child with CF.",
insights:"Joshua showed us just how dire the situation is for CF patients is in some regions. It was shocking to hear there is only one doctor knowledgeable about CF in Thailand and that many doctors dismiss the possibility of CF due to racial bias and misinformation. Additionally, we confirmed how much the accessibility of care depends on the healthcare system, as we already touched on during the interview with Nicole Friedlein [link]. On the parenting level, Joshua brought in many perspectives contrary to what we previously heard. In the interview with Max [Link], we learned he vehemently avoids ponding water while Joshua’s daughter is allowed to roam around with no such restrictions. Neither have chronic infections. ",
insights:"Joshua showed us just how dire the situation is for CF patients is in some regions. It was shocking to hear there is only one doctor knowledgeable about CF in Thailand and that many doctors dismiss the possibility of CF due to racial bias and misinformation. Additionally, we confirmed how much the accessibility of care depends on the healthcare system, as we already touched on during the interview with Nicole Friedlein [link]. On the parenting level, Joshua brought in many perspectives contrary to what we previously heard. In the interview with Max [Link], we learned he vehemently avoids ponding water while Joshua’s daughter is allowed to roam around with no such restrictions. Neither have chronic infections. ",
implementation:"The interview with Josh made us realize we too needed to look at the reason why we chose F508del. Did we, too, fall for bias? Despite a change of target not being feasible anymore, we looked into it and traced back our steps that led to our decision. We did not find as much information about other mutations when first researching cystic fibrosis, especially in the context of prime editing. Mattijs Bulceans's paper on targeting the mutations L227R and N1303K [1] was one of few papers. After explicitly searching for cystic fibrosis records for specific countries and regions, we uncovered a moderate number of papers examining CF in Asia and other regions we previously did not know much about. The very first article supported Joshua's hypotheses and painted a sad picture: Among other things, it describes the case of a four-month-old boy who was diagnosed with cystic fibrosis. Nothing unusual in itself, but the circumstances are depressing. Two of the three siblings born before him died within months of birth and had previously presented with symptoms of cystic fibrosis. He was the first to be diagnosed. A sweat test aimed at cystic fibrosis was not available at the hospital, so one was improvised. Later on, a genetic test revealed the presence of 508del. [2] We found ourselves and our lack of knowledge in good company as we found papers as new as from 2020 (14 years after the previously mentioned paper) containing statements such as “recent reports suggest that CF does occur in Asia” [3]. Fortunately, there is a rising number of cystic fibrosis experts for Asia and other previously overlooked regions such as Africa. [4] We chose to not only look at the scientific data but also into anecdotal evidence. To find the latter, we searched official and private websites and chatrooms for information and experiences of patients. In the end, we found narratives from most ethnic backgrounds about being dismissed and often misdiagnosed. Of course, this is not an occurrence unique to cystic fibrosis. Our conclusion is that yes, we did fall for bias. But regardless of ethnicity, 508del occurs and is overall the most prevalent mutation as was confirmed in our interview with CF expert Sriram .... This experience was uncomfortable as we felt the pressure to be thorough and deliver a perfect project. What would have been more devastating than realizing we made a wrong choice at the very core? We made the conscious decision to invest our resources into figuring out if we indeed made a mistake and we want to encourage other teams to do the same. iGem stands for innovation – but also for growth. Especially in the context of Integrated Human Practices, it is important to examine both the positive and the negative to create a project with a future. ",
implementation:"The interview with Josh made us realize we too needed to look at the reason why we chose F508del. Did we, too, fall for bias? Despite a change of target not being feasible anymore, we looked into it and traced back our steps that led to our decision. We did not find as much information about other mutations when first researching cystic fibrosis, especially in the context of prime editing. Mattijs Bulceans's paper on targeting the mutations L227R and N1303K [1] was one of few papers. After explicitly searching for cystic fibrosis records for specific countries and regions, we uncovered a moderate number of papers examining CF in Asia and other regions we previously did not know much about. The very first article supported Joshua's hypotheses and painted a sad picture: Among other things, it describes the case of a four-month-old boy who was diagnosed with cystic fibrosis. Nothing unusual in itself, but the circumstances are depressing. Two of the three siblings born before him died within months of birth and had previously presented with symptoms of cystic fibrosis. He was the first to be diagnosed. A sweat test aimed at cystic fibrosis was not available at the hospital, so one was improvised. Later on, a genetic test revealed the presence of 508del. [2] We found ourselves and our lack of knowledge in good company as we found papers as new as from 2020 (14 years after the previously mentioned paper) containing statements such as “recent reports suggest that CF does occur in Asia” [3]. Fortunately, there is a rising number of cystic fibrosis experts for Asia and other previously overlooked regions such as Africa. [4] We chose to not only look at the scientific data but also into anecdotal evidence. To find the latter, we searched official and private websites and chatrooms for information and experiences of patients. In the end, we found narratives from most ethnic backgrounds about being dismissed and often misdiagnosed. Of course, this is not an occurrence unique to cystic fibrosis. Our conclusion is that yes, we did fall for bias. But regardless of ethnicity, 508del occurs and is overall the most prevalent mutation as was confirmed in our interview with CF expert Sriram .... This experience was uncomfortable as we felt the pressure to be thorough and deliver a perfect project. What would have been more devastating than realizing we made a wrong choice at the very core? We made the conscious decision to invest our resources into figuring out if we indeed made a mistake and we want to encourage other teams to do the same. iGem stands for innovation – but also for growth. Especially in the context of Integrated Human Practices, it is important to examine both the positive and the negative to create a project with a future. ",
