<p>The transfection efficiency was much better. Our proof-of-concept was working correctly. The reporter system pDAS12189 only led to production of a fluorescent signal when co transfected with a prime editing complex as pCMV-PE2.</p>
</Subesction>
<Subesctiontitle="Mechanism"id="Results2">
<H4text="Goals"/>
<p>text</p>
<H4text="Workflow"/>
<p>text</p>
<H4text="Conclusion"/>
<p>text</p>
<H4text="pegRNA 1"/>
<H5text="Workflow"/>
<p>With this experiment we wanted to compare the efficiency of pegRNAs with and without silent edits.</p>
<H5text="Conclusion"/>
<p>The FACS analysis shows that pegRNA without silent edits (pegRNA1) had a 2.05 times higher transfection efficiency than pegRNA with silent edits (pegRNA2).</p>
<H4text="pegRNA 2"/>
<H5text="Workflow"/>
<p>Cotransfection of pPEAR_CFTR and PE2 and also 1 of the 14 pegRNAs to compare the transfection efficiency of all of our designed pegRNAs.</p>
<H5text="Conclusion"/>
<p>The pegRNAs lead to differing amounts of cells showing fluorescence, which, assuming comparable transfection efficiencies, indicates varying prime editing efficiency. The pegRNA7 showed the highest transfection efficiency (see Figure X).</p>
<H4text="pegRNA 3"/>
<H5text="Workflow"/>
<p>We tried to transfect CFBE41o- cells with pDAS12124-preedited, our internal positive control, to check if a transfection of this cell line is possible. Furthermore we tried to co transfect the CFBE41o- with pPEAR_CFTR, PE6c and pegRNA4.</p>
<H5text="Conclusion"/>
<p>Transfection of CFBE41o- with pDAS12124-preedited was successful (see Figure X). After 24 hours a successful co transfection of pPEAR_CFTR with PE6c and pegRNA4 was visible, although the transfection efficiency was really bad (see Figure X).</p>
<H4text="pegRNA 4"/>
<H5text="Workflow"/>
<p>pCMV-PE2 was co transfected with pDAS12489 and pCMV-PE_CO-Mini was co transfected with pDAS12489 in HEK293 cell line.</p>
<H5text="Conclusion"/>
<p>The FACS results show that transfection with pCMV-PE2 as the prime editing complex had editing efficiency of 52.90% when normalized on pDAS12124-preedited. When pCMV-PE_CO-Mini was used as a prime editing complex it had a transfection efficiency of 2.54% (see Figure X).</p>
<H4text="pegRNA 5"/>
<H5text="Workflow"/>
<p>We compared the 3 different Prime Editing complexes (pCMV-PE2, pCMV-PE2_CO-Mini & pCMV-PE6c) to check which one has the best transfection efficiency.</p>
<H5text="Conclusion"/>
<p>The FACS measurement shows the fluorescence rate cells co-transfected with pDAS12489 and pCMV-PE6c as a prime editing complex. The editing efficiency off PE6c was by far the highest (81.88%) (see Figure X). The efficiency was 1.55 higher than the efficiency when pCMV-PE2 was used as prime editing complex (see Figure X).</p>