Difference between revisions of "Part:BBa K4830009"

 
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<partinfo>BBa_K4830009 short</partinfo>
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POAG snp1-3 ngRNA - nicking guide RNA targeting the Single nucleotide polymorphism causing Primary open-angle glaucoma 
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===Usage and Biology===
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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.
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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.
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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.
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PE3b has an additional single guide (sgRNA) that nicks the unedited strand at a location away from the editing site. PE3b sgRNAs are designed with spacers to match the edited strand but not the original allele. By doing this, mismatches between the spacer and the unedited allele should disfavor sgRNA nicking until after editing of the PAM strand has occurred.
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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.
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===Characterization===
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The ngRNA in combination with pegRNA was used to test the efficiency of the alternative RT in comparison to controls.
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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 ngRNA design.
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<img src = "https://static.igem.wiki/teams/4830/wiki/poag.png" style = "width:440px;height:380px">
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Fig. 1 Prime editing activity of PE2 at POAG snp1-3 target site.
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4830009 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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<partinfo>BBa_K4830009 parameters</partinfo>
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Latest revision as of 11:07, 12 October 2023


POAG snp1-3 ngRNA

POAG snp1-3 ngRNA - nicking 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. PE3b has an additional single guide (sgRNA) that nicks the unedited strand at a location away from the editing site. PE3b sgRNAs are designed with spacers to match the edited strand but not the original allele. By doing this, mismatches between the spacer and the unedited allele should disfavor sgRNA nicking until after editing of the PAM strand has occurred.

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 ngRNA in combination with pegRNA 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 ngRNA design.

Fig. 1 Prime editing activity of PE2 at POAG snp1-3 target site.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]