<Subesctiontitle="Adaptions for CFTR F508del mutation comparibility"id="Characterization2">
<p>In addition to the introduction of the three-base-pair deletion, the intron sequence was further altered with the objective of enhancing the comparability of the system to the CFTR genomic context. Specifically, 27 base pairs were replaced downstream of the splice site with a sequence derived from the CFTR gene in the region of the F508del mutation. This modification guarantees that the gRNA spacer employed in our system is identical to the one found in the actual genomic context of the CFTR mutation. </p>
<p>The only notable differences between the system and the genomic sequence are observed in the RTT (Reverse Transcript Template) and PBS (Primer Binding Site), which have been calibrated with silent mutations to maintain comparability in GC content with the native CFTR gene. These silent mutations do not affect the encoded protein but optimise the system's mimicry of the CFTR gene. </p>
<p>Subsequently, a purification process is conducted to extract the plasmid backbone and concentrate the samples. This facilitates the integration of the amplified reporter system into the prepared pDAS12124_PEAR-GFP-preedited backbone, which is then subjected to the Gibson assembly process. This assembly process results in the creation of the novel pDAS12124_PEAR-GFP_GGTdel_edited plasmid, which incorporates the CF-specific reporter system. </p>
<p>Subsequently, the pDAS12124_PEAR-GFP_GGTdel_edited plasmid is transformed into E. coli DH5α cells for propagation. To confirm the successful integration of the reporter fragment, colony PCR (cPCR) is performed on the transformed colonies. The positive colonies, identified by cPCR, are selected and grown in LBCm50 medium for further analysis. </p>
<p>The final validation step involves preparing the pDAS12124_PEAR-GFP_GGTdel_edited plasmid from the positive colonies and verifying the correct insertion of the reporter fragment using Sanger sequencing. This ensures the fragment is inserted in the correct orientation and that the CF-specific reporter system has been successfully constructed without any errors. </p>
<p>In connection with the optimisation of prime editing with regard to the F508del mutation, it was necessary to compare different pegRNAs, as their optimal structure always depends on the application context. We therefore designed and cloned 14 variants of pegRNAs for the target of the reporter system and then tested them on the reporter system using the PE2 system. </p>
<p>For pegRNA screening, we co-transfected the HEK293 cells with our modified reporter plasmid, the pegRNA expressing plasmid and pCMV-PE2. We were then able to measure the fluorescence after 72 hours using FACS and evaluate which pegRNA showed the highest efficiency. </p>
<figcaption><b>Figure 9.</b>Percentage of fluorescent HEK293 cells 72 h after transfection with various pegRNAs (pegRNA1-14) normalized to pDAS12124 pre-edited as internal positive control as result of flow cytometry analysis.</figcaption>
description="Percentage of fluorescent HEK293 cells 72 h after transfection with various pegRNAs (pegRNA1-14) normalized to pDAS12124 pre-edited as internal positive control as result of flow cytometry analysis"
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<p>We also co-transfected the CFBE41o- with our modified reporter plasmid, the plasmid expressing pegRNA04 as well as pCMV-PE6c. As a result, we observed fluorescence, indicating successful editing of the reporter plasmid. The negative controls transfected with only one of the plasmids each showed no fluorescence, routing out other factors. This gave us validation, that our pegRNAs work not only in HEK, but also in epithelial cells that express CFTR F508del. </p>
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description="Microscopy results after 24h or 48h. Transfection of pDAS12124-preedited with lipofectamine 3000 was successfully done in CFBE41o- cell line and visible after 48h. CFBE41o- cell line was transfected with pDAS-IDT with Lipofectamine 3000 and afterwards with LNPs including PE6c and pegRNA4 and was after 24h fluorescence visible."
Biosafety is also guaranteed by the careful selection of the spacer, which plays a critical role in guiding the complex to its intended target site <SupScrollLinklabel="2"/>. To ensure both precision and safety, we meticulously chose and rigorously checked the spacer using the <ahref="https://www.synthego.com/products/bioinformatics/crispr-design-tool">CRISPick software</a><SupScrollLinklabel="3"/>. This allowed us to evaluate whether our Spacer would be likely to target other regions than our target site and therefore allowing us to analyse and predict potential off-target effects, ensuring that erroneous edits are minimised. By optimising the spacer selection, we have not only significantly enhanced the overall editing efficiency, striking a balance between precision and performance, but especially ensured the utmost accuracy in directing the Prime Editor, further contributing to the safety of the editing process.
description="Illustration of the introduction of silent mutations leading to the PAM disrupt"
alt1="Illustration of the introduction of silent mutations leading to the PAM disrupt"
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<H4text="Riboswitch"></H4>
<p>
Riboswitches are segments of an RNA strand that bind to small molecules, causing them to change their secondary structure by forming hairpin structures. This process regulates gene expression at the translation level by preventing ribosomes from binding at the RBS and translating the coding region on the RNA strand. 0For our project we also considered an ion-sensitive riboswitch, specifically dependent on sodium ions (Na⁺), as a regulatory mechanism. The secondary structure of this riboswitch prevents the binding of ribosomes to the ribosome binding site (RBS) under normal conditions, thus inhibiting the translation of the subsequent mRNA. When sodium ions bind to the riboswitch, a structural change occurs, exposing the RBS, which allows for the translation of the mRNA and the production of our fusion protein which is the main component of our prime editing system and therefore of enormous importance for it to work <SupScrollLinklabel="4"/>. In the context of the CFTR mutation and its effects on the cell, the elevated Na⁺ levels play a crucial role. Due to the dysfunctional CFTR channel, which fails to properly function as a chloride channel, the ENaC channel (epithelial sodium channel) becomes upregulated. This upregulation results in an increased transport of sodium ions into the cell, leading to a higher intracellular sodium concentration. This elevated Na⁺ concentration creates a specific ionic environment that could potentially be utilized to regulate our Prime-Editing complex in a targeted manner. Given these specific ionic changes in the cell, we could have a disease-specific regulation of our Prime-Editing system based on the ionic situation typical of this condition. However, despite the initial promise of this approach, after further research, we concluded that the riboswitch, even considering the ion levels within epithelial cells, is overall too nonspecific and therefore too unreliable as a regulatory mechanism. Although the ion levels in CFTR cells are much lower, there are still low concentrations of sodium ions, which can lead to the riboswitch not being completely switched off.
description="Illustration of the mechanism of action of the riboswitch"
alt1="Illustration of the mechanism of action of the riboswitch"
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<p>
As a further approach to developing alternative riboswitch variants, we considered the possibility of an RNA-regulated riboswitch targeting the defective mRNA sequence of the genetically defective CFTR gene. The basic idea behind this concept was that the riboswitch specifically binds to a region on the CFTR mRNA containing the F508Δ mutation. This binding should induce a structural change in the riboswitch on our prime editing complex’s mRNA that ultimately leads to exposure of the RBS to allow translation of the downstream sequence. This mechanism would be designed to react specifically to the defective CFTR mRNA and only cause a change in the secondary structure in the presence of the specific mutation. The riboswitch could thus ensure selective and disease-specific activation of our prime editing complex, which would be of particular interest in the context of genetic diseases such as cystic fibrosis. However, we did not pursue this approach any further. A major reason for this was the lack of sufficient literature providing a sound scientific basis for this specific application of a riboswitch. In
addition, our research steered us in a different direction, particularly with regard to the alternative mechanism involving the XBP1 intron to regulate the prime editing system. This alternative seemed more promising and was based on an established regulatory mechanism that is triggered by cellular stress and specifically responds to misfolding processes.
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.
<figcaption><b>Figure 1</b>Photo-gallery of laboratory. A: Key lock. B: Key-locked door. C: Alarm plan. D: Emergeny button for electriotion stop. E: Emergency telephone. F: First aid kit, cardiac defibrillaton and emergency exit and fire alarm plan. G: Wash bin with emergency eye wash. H: Emergency shower. I: Lockable cabinets for chemical storage. </figcaption>
<figcaption><b>Figure 2</b>Photo-gallery of S1 laboratory. A: Autoclave. B: Refrigerator with chemicals. C: Weighing room with chemical storage. D: Clean bench work space with vortex, pipettes, heat block and bench top centrifuge. E: pH electrode in fume hood. F: Ice machine. G: Fire distinguisher and S1 waste. H: Fume hood with liquid waste.</figcaption>
description="Photo-gallery of laboratory. A: Key lock. B: Key-locked door. C: Alarm plan. D: Emergeny button for electriotion stop. E: Emergency telephone. F: First aid kit, cardiac defibrillaton and emergency exit and fire alarm plan. G: Wash bin with emergency eye wash. H: Emergency shower. I: Lockable cabinets for chemical storage"
alt1="Photo-gallery of laboratory. A: Key lock. B: Key-locked door. C: Alarm plan. D: Emergeny button for electriotion stop. E: Emergency telephone. F: First aid kit, cardiac defibrillaton and emergency exit and fire alarm plan. G: Wash bin with emergency eye wash. H: Emergency shower. I: Lockable cabinets for chemical storage"
description="Photo-gallery of S1 laboratory. A: Autoclave. B: Refrigerator with chemicals. C: Weighing room with chemical storage. D: Clean bench work space with vortex, pipettes, heat block and bench top centrifuge. E: pH electrode in fume hood. F: Ice machine. G: Fire distinguisher and S1 waste. H: Fume hood with liquid waste"
alt1="Photo-gallery of S1 laboratory. A: Autoclave. B: Refrigerator with chemicals. C: Weighing room with chemical storage. D: Clean bench work space with vortex, pipettes, heat block and bench top centrifuge. E: pH electrode in fume hood. F: Ice machine. G: Fire distinguisher and S1 waste. H: Fume hood with liquid waste."
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</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.
description="Photo-gallery of laboratory and chemical storage. A: Safety cabinets. B: Incubator. C: Safety cabinet"
alt1="Photo-gallery of laboratory and chemical storage. A: Safety cabinets. B: Incubator. C: Safety cabinet"
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<p>
In our S2 laboratory, the harvested nasal epithelial cells that serve as primary cultures undergo a comprehensive HHH test 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.
