Part:BBa_K4830006

HEK3 pegRNA

HEK3 pegRNA - prime editing guide RNA targeting the gene encoding EPHA8 (EPH Receptor A8), and introduces CTT insertion

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.

EPHA8 (EPH Receptor A8) is a gene encoding a member of the ephrin receptor subfamily of the protein-tyrosine kinase family. EPH and EPH-related receptors participate in development of the mammalian nervous system.

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, in this case CTT nucleoide sequence insertion at the HEK3 target site. 3 plasmids containing PE, pegRNA and ngRNA were trasfected into HEK293T cells, and editing efficiency was evaluated 72hr after transfection using Sanger sequencing. Figure 1 shows the editing efficiency of the alternative RTs on RNF2 target site, proving the validity of the pegRNA design.

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]