Part:BBa_K4830026

777BFP ngRNA

777BFP ngRNA - nicking guide RNA (ngRNA) targeting the premature stop codon instilled in an eGFP. The 777BFP ngRNA is used concurrently with the TLR-76 pegRNA in an 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. It has an excitation maxima of 488nm, and emission maxima of 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 the percentage of cells containing the eGFP.

TLR-76 pegRNA (BBa_K4830025) was co-transfected with 777BFP ngRNA (BBa_K4830026). The targeting sequence was the premature stop codon found on the eGFP requiring a complex edit (BBa_K4830029). Initial testing were performed on two different cell lines containing the integrated sequence. HEK293T TLR WT is a heterogeneous cell line containing the integrated sequence, while HEK293T 1D10 is a homogeneous cell line derived from the former.

Fig. 1 Bar graph shows the P3 FITC-H Mean (indicating the mean fluorescence intensity of eGFP), and scatter points denote the P3% Parent (indicating the percentage of cell containing correctly edited wild-type eGFP). Results from initial testing with TLR WT cell line.

Fig. 2 Bar graph shows the P3 FITC-H Mean (indicating the mean fluorescence intensity of eGFP), and scatter points denote the P3% Parent (indicating the percentage of cell containing correctly edited wild-type eGFP). Results from initial testing with TLR 1D10 cell line.

Sequence and Features

The 777BPF ngRNA sequence includes the spacer and gRNA scaffold.

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]