<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="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.
</p>
<p>
<strong>pegRNA (Prime Editing Guide RNA):</strong> The pegRNA is a multifunctional RNA molecule that fulfils two essential tasks. Firstly, it serves as a standard guide RNA (gRNA) that binds specifically to the target DNA and thus marks the site of editing. Secondly, it contains an RNA template that encodes the desired DNA modification. This enables the precise integration of the genetic modifications at the target site. We evaluated pegRNA for its ability to specifically target and modified the intended DNA sequence. Ensuring its specificity was crucial to avoid the potential disruption of other genes.
</p>
<p>
<strong>Nickase nCas9, CasX, Fanzor (SpuFz1):</strong> These modified nucleases are designed to cut only one strand of DNA. This leads to controlled and precise editing of the genome, as cutting only one strand minimizes the risk of unwanted double-strand breaks. CasX and Fanzor offer smaller alternatives to Cas9, which is particularly advantageous for use in cells or organisms where space and efficiency requirements in terms of the transport system are an issue. Fanzor, being a newly introduced endonuclease, was particularly scrutinized in our project to ensure its safety and effectiveness in different cellular contexts.
<Collapsibleid="Checkpek"open={false}title="see full article here">
<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.
</p>
<p>
<strong>pegRNA (Prime Editing Guide RNA):</strong> The pegRNA is a multifunctional RNA molecule that fulfils two essential tasks. Firstly, it serves as a standard guide RNA (gRNA) that binds specifically to the target DNA and thus marks the site of editing. Secondly, it contains an RNA template that encodes the desired DNA modification. This enables the precise integration of the genetic modifications at the target site. We evaluated pegRNA for its ability to specifically target and modified the intended DNA sequence. Ensuring its specificity was crucial to avoid the potential disruption of other genes.
</p>
<p>
<strong>Nickase nCas9, CasX, Fanzor (SpuFz1):</strong> These modified nucleases are designed to cut only one strand of DNA. This leads to controlled and precise editing of the genome, as cutting only one strand minimizes the risk of unwanted double-strand breaks. CasX and Fanzor offer smaller alternatives to Cas9, which is particularly advantageous for use in cells or organisms where space and efficiency requirements in terms of the transport system are an issue. Fanzor, being a newly introduced endonuclease, was particularly scrutinized in our project to ensure its safety and effectiveness in different cellular contexts.
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>
</Collapsible>
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="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, 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 check-In and the specific safety measures:
