Part:BBa_K4830001

RNF ngRNA

RNF2 ngRNA - nicking guide RNA targeting the gene encoding Ring Finger Protein 2 (RNF)

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. PE3b has an additional single guide (sgRNA) that nicks the unedited strand at a location away from the editing site. PE3b sgRNAs are designed with spacers to match the edited strand but not the original allele. By doing this, mismatches between the spacer and the unedited allele should disfavor sgRNA nicking until after editing of the PAM strand has occurred.

RNF2 also known as Ring Finger Protein 2 is a gene encoding for protein, that is part of Polycomb group (PcG) of proteins. This complex is significant in the transcription repression of various genes involved in development and cell proliferation. Luo-Schoch-Yamamoto Syndrome and Non-Specific Syndromic Intellectual Disability are associated with RNF2.

Characterization

The pegRNA in combination with ngRNA were used to test the efficiency of the Prime Editor containing the alternative Reverse Trancriptases. The pegRNA serves as template to install the edit, in this case base conversion from A to G (T to C) at the RNF2 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 ngRNA design.

Fig. 1 Screening of RT variants for prime editing activity at RNF2 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]