Difference between revisions of "Part:BBa K4830024"

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−	Fig. 1 Bar graph shows the mean P2 FITC-A from the three replicates, and scatter points denote the individual P2 FITC-A from each replicate.	+	Fig. 1 Bar graph shows the mean P2 FITC-A from the three replicates, and scatter points denote the individual P2 FITC-A from each replicate. P2 FITC-A is a measurement of the mean fluorescence intensity of the GFP.
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Revision as of 11:51, 12 October 2023

PETLR ngRNA 1

PETLR ngRNA 1 - nicking guide RNA (pegRNA) targeting the premature stop codon instilled in an eGFP. The PETLR ngRNA 1 is used in the improved Traffic Light Reporter (TLR) assay for prime editing adapted from the TLR paper in the References.

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.

eGFP is a green fluorescent protein derived from Aequorea victoria. eGFP has an excitation wavelength peak of 488nm and emission wavelength peak at 509nm.

Characterization

The pegRNA in combination with ngRNA were used to test the efficiency of the Prime Editor containing the alternative Reverse Trancriptases. The pegRNA served as template to install the edit. 3 plasmids containing PE, pegRNA and ngRNA were trasfected into HEK293T cells, and editing efficiency was evaluated 72hr after transfection using flow cytometry by evaluating either the mean fluorescence intensity of the green fluorescence protein (GFP), or percentage of cells containing the GFP.

PETLR pegRNA 1 (BBa_K4830023) was co-transfected with PETLR ngRNA 1 (BBa_K4830024). The targeting sequence was the premature stop codon found on the eGFP requiring a simple edit (BBa_K4830028).

Fig. 1 Bar graph shows the mean P2 FITC-A from the three replicates, and scatter points denote the individual P2 FITC-A from each replicate. P2 FITC-A is a measurement of the mean fluorescence intensity of the GFP.

Sequence and Features

The sequence contains the U6 promoter, spacer and gRNA scaffold.