Difference between revisions of "Part:BBa K4830008"
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To initiate prime editing, PE creates a single-strand break in the DNA at the target site to allow reverse transcriptase to access the DNA and synthesize a new DNA strand using pegRNA as a template. Then the information from the edited strand is copied to the complementary strand through the cell’s natural repair pathways. | To initiate prime editing, PE creates a single-strand break in the DNA at the target site to allow reverse transcriptase to access the DNA and synthesize a new DNA strand using pegRNA as a template. Then the information from the edited strand is copied to the complementary strand through the cell’s natural repair pathways. | ||
| − | Single nucleotide polymorphism is variation at a single base position in DNA, and in this case associated with POAG, leading cause of blindness, characterized by optic nerve degeneration. | + | Single nucleotide polymorphism is variation at a single base position in DNA, and in this case associated with POAG, leading cause of blindness, characterized by optic nerve degeneration. It is one of the therapeutic sites to test Prime Editing on. |
===Characterization=== | ===Characterization=== | ||
The pegRNA in combination with ngRNA was used to test the efficiency of the alternative RT in comparison to controls. | The pegRNA in combination with ngRNA was used to test the efficiency of the alternative RT in comparison to controls. | ||
| − | 3 plasmids containing PE, pegRNA and ngRNA were trasfected into HEK293T cells, and editing efficiency was evaluated 72hr after transfection using Sanger sequencing. Figure 1 shows the editing efficiency of the original PE2 and truncated PE2 on | + | 3 plasmids containing PE, pegRNA and ngRNA were trasfected into HEK293T cells, and editing efficiency was evaluated 72hr after transfection using Sanger sequencing. Figure 1 shows the editing efficiency of the original PE2 and truncated PE2 on POAG snp1-3 target site, proving the validity of the pegRNA design. |
<html> | <html> | ||
<div class = "middle"> | <div class = "middle"> | ||
| − | <img src = "https://static.igem.wiki/teams/4830/wiki/poag.png" style = "width: | + | <img src = "https://static.igem.wiki/teams/4830/wiki/poag.png" style = "width:440px;height:380px"> |
</div> | </div> | ||
</html> | </html> | ||
| − | Fig. 1 Prime editing activity of PE2 at | + | Fig. 1 Prime editing activity of PE2 at POAG snp1-3 target site. |
Latest revision as of 11:06, 12 October 2023
POAG snp1-3 pegRNA
POAG snp1-3 pegRNA - prime editing guide RNA targeting the Single nucleotide polymorphism causing Primary open-angle glaucoma
Usage and Biology
Prime editing is an innovative technology for genome editing that enables the installation of the wide spectrum of gene modifications such as 12 possible base-to-base conversions, small insertions, and deletions, without requiring double-stranded breaks or donor DNA templates. This technology provides high versatility and target specificity, offering the potential to revolutionize medicine by providing novel tools for treating genetic diseases.
Prime editing relies on specialized prime editors, which usually consist of reverse transcriptase enzyme fused to nickase Cas9, and prime editing guide RNA containing a spacer that specifies the target site. It also includes a scaffold and 3’ extension containing a primer binding site (PBS) and an RT template encoding the desired edit. To initiate prime editing, PE creates a single-strand break in the DNA at the target site to allow reverse transcriptase to access the DNA and synthesize a new DNA strand using pegRNA as a template. Then the information from the edited strand is copied to the complementary strand through the cell’s natural repair pathways.
Single nucleotide polymorphism is variation at a single base position in DNA, and in this case associated with POAG, leading cause of blindness, characterized by optic nerve degeneration. It is one of the therapeutic sites to test Prime Editing on.
Characterization
The pegRNA in combination with ngRNA was used to test the efficiency of the alternative RT in comparison to controls. 3 plasmids containing PE, pegRNA and ngRNA were trasfected into HEK293T cells, and editing efficiency was evaluated 72hr after transfection using Sanger sequencing. Figure 1 shows the editing efficiency of the original PE2 and truncated PE2 on POAG snp1-3 target site, proving the validity of the pegRNA design.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]