Difference between revisions of "Part:BBa K4830015"
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<partinfo>BBa_K4830015 short</partinfo> | <partinfo>BBa_K4830015 short</partinfo> | ||
| − | Prototype Foamy virus Reverse transcriptase. | + | PFV RT is Prototype Foamy virus Reverse transcriptase. |
<!-- Add more about the biology of this part here --> | <!-- Add more about the biology of this part here --> | ||
===Usage and Biology=== | ===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. | ||
| + | |||
| + | Reverse transcriptases (RTs) are RNA-dependent DNA polymerases isolated from retroviruses, used to generate complementary DNA (cDNA) from an RNA template in process of reverse transcription. | ||
| + | |||
| + | Prototype foamy virus (PFV) is a member of the foamy viruses (FVs; also known as spumaviruses) belonging to a subfamily of the Retroviridae family. | ||
===Characterization=== | ===Characterization=== | ||
| − | + | Reverse Trancriptase fused to nickase Cas9 BBa K4830014, in plasmid form was co-transfected with pegRNA and ngRNA targeting different endogeneous genomic loci on HEK293T cells and the editing efficiency was estimated after sanger sequencing. Fig. 1, Fig. 2 and Fig.3 show the editing efficiency of PFV RT along with other the alternative RTs on RNF2, HEK3 and EMX1 target sites respectively. | |
<html> | <html> | ||
<div class = "middle"> | <div class = "middle"> | ||
| − | <img src = "https://static.igem.wiki/teams/4830/wiki/hek3- | + | <img src = "https://static.igem.wiki/teams/4830/wiki/hek3-alt-rt.png" style = "width:650px;height:500px"> |
| − | + | ||
</div> | </div> | ||
| − | </html> | + | Fig. 1 Screening of RT variants for prime editing activity at HEK3 target site. Bar graphs show the mean value and error bars indicate S.D. of n = 3, independent biological replicates. |
| + | </div> | ||
| + | <img src = "https://static.igem.wiki/teams/4830/wiki/rng2-alt-rt.png" style = "width:650px;height:500px"> | ||
| + | </div> | ||
| + | Fig. 2 Screening of RT variants for prime editing activity at RNF2 target site. Bar graphs show the mean value and error bars indicate S.D. of n = 3, independent biological replicates. | ||
| + | </div> | ||
| + | <img src = "https://static.igem.wiki/teams/4830/wiki/emx1-alt-rt.png" style = "width:650px;height:500px"> | ||
| + | </div> | ||
| + | |||
| + | </html> | ||
| + | Fig. 3 Screening of RT variants for prime editing activity at EMX1 target site. | ||
Latest revision as of 02:33, 12 October 2023
PFV RT
PFV RT is Prototype Foamy virus Reverse transcriptase.
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.
Reverse transcriptases (RTs) are RNA-dependent DNA polymerases isolated from retroviruses, used to generate complementary DNA (cDNA) from an RNA template in process of reverse transcription.
Prototype foamy virus (PFV) is a member of the foamy viruses (FVs; also known as spumaviruses) belonging to a subfamily of the Retroviridae family.
Characterization
Reverse Trancriptase fused to nickase Cas9 BBa K4830014, in plasmid form was co-transfected with pegRNA and ngRNA targeting different endogeneous genomic loci on HEK293T cells and the editing efficiency was estimated after sanger sequencing. Fig. 1, Fig. 2 and Fig.3 show the editing efficiency of PFV RT along with other the alternative RTs on RNF2, HEK3 and EMX1 target sites respectively.
