Part:BBa_K4830012

FANCF pegRNA

FANCF pegRNA - prime editing guide RNA targeting the gene encoding FA Complementation Group F, and introduces G to T (C to A) nucleotide change

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.

FANCF (FA Complementation Group F) is gene encoding protein, associated with Fanconi Anemia. This disease is characterized by defective DNA repair, increased chromosomal breakage and cytogenetic instability.

Characterization

The pegRNA in combination with ngRNA were used to test the efficiency of the Prime Editor 2. The pegRNA serves as template to install the edit, in this case G to T (C to A) at the FANCF target site. 3 plasmids containing PE2, 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 original PE2 and truncated PE2 on FANCF target site, proving the validity of the pegRNA design.

Fig. 1 Prime editing activity of PE2 at FANCF target site.

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]