Part:BBa_K4830023

PETLR pegRNA 1

PETLR pegRNA 1 - prime editing guide RNA (pegRNA) targeting the premature stop codon instilled in an eGFP; The premature stop codon is edited into the translatable amino acid - threonine, restoring the wild-type eGFP sequence. The PETLR pegRNA 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 enhanced green fluorescent protein (eGFP), or percentage of cells containing the eGFP.

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 eGFP.

Sequence and Features

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

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]