Difference between revisions of "Part:BBa K4830017"
(→‎Characterization)
 
(4 intermediate revisions by the same user not shown)
Line 3: Line 3:
 
<partinfo>BBa_K4830017 short</partinfo>
 
<partinfo>BBa_K4830017 short</partinfo>
  
−
Ll.ltrA reverse transcriptase is one of proteins found in The Lactococcus lactis Ll.LtrB intron, that binds the intron RNA to promote RNA splicing and intron mobility.
+
Ll.ltrA RT group II intron 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===
  
−
Ll.ltrA was one of the Reverse Transcriptases we attempted to use as alternative RT for Prime editing.  
+
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.
 +
 
 +
Ll.ltrA reverse transcriptase is one of proteins found in The Lactococcus lactis Ll.LtrB intron, that binds the intron RNA to promote RNA splicing and intron mobility.  
  
 
===Characterization===
 
===Characterization===
  
−
To evaluate the performance of various Ll.ltrA, we cotransfected the RT with pegRNA and ngRNA targeting the RNF and HEK3 genomic loci on HEK293T cells and estimated the editing efficiency using sanger sequencing. Another evaluation test was performed on TLR reporter system at 24hr and 72hr transfection time.The results are plotted as bar graph shown below. Comparison was done against the wild-type PE2 and PE2 with truncated RNAse H region (PE-497).
+
Reverse Trancriptase fused to nickase Cas9 BBa K4830014, in plasmid form was co-transfected with pegRNA and ngRNA targeting endogeneous genomic loci on HEK293T cells and the editing efficiency was estimated after sanger sequencing. Figure 1 shows the editing efficiency of Ll.ltrA RT along with other the alternative RTs on HEK3 target site.
 +
 
 +
<html>
 +
<div class = "middle">
 +
<img src = "https://static.igem.wiki/teams/4830/wiki/hek3-alt-rt.png" style = "width:650px;height:500px">
 +
</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.
  
 
<!-- -->
 
<!-- -->

Latest revision as of 02:26, 12 October 2023


Ll.ltrA RT

Ll.ltrA RT group II intron 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.

Ll.ltrA reverse transcriptase is one of proteins found in The Lactococcus lactis Ll.LtrB intron, that binds the intron RNA to promote RNA splicing and intron mobility.

Characterization

Reverse Trancriptase fused to nickase Cas9 BBa K4830014, in plasmid form was co-transfected with pegRNA and ngRNA targeting endogeneous genomic loci on HEK293T cells and the editing efficiency was estimated after sanger sequencing. Figure 1 shows the editing efficiency of Ll.ltrA RT along with other the alternative RTs on HEK3 target site.

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.

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]