Part:BBa_K4830021

TLR pegRNA 2

TLR pegRNA 2 - prime editing guide RNA (pegRNA) targeting the premature stop codon instilled in a mCherry fluorescent protein (mCherry); introduces A to C (T to G) nucleotide change. The premature stop codon is edited into the translatable amino acid - glycine, restoring the wild-type mCherry sequence. The TLR pegRNA 2 is used in the 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.

mCherry is a red fluorescent protein derived from Discosoma sp. mCherry has an excitation wavelength peak of 587nm and emission wavelength peak at 610nm.

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 fluorescent protein (GFP), or percentage of cells containing the GFP.

TLR pegRNA 1 (BBa_K4830019) was co-transfected with TLR ngRNA 1 (BBa_K4830020); while TLR pegRNA 2 (BBa_K4830021) was co-transfected with TLR ngRNA 2 (BBa_K4830021). The targeting sequence was the premature stop codon found on the mCherry protein (BBa_K4830027).

Fig. 1 Bar graph shows the mean fluorescence intensity (MFI) for GFP, and scatter points denote the %GFP cells in one of the replicates of the initial TLR assay.

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]