Part:BBa_K4830010

ADAR pegRNA

ADAR pegRNA - prime editing guide RNA targeting the gene encoding Adenosine Deaminase RNA Specific, introducing T to A, and A to C nucleotide changes.

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.

ADAR (Adenosine Deaminase RNA Specific) is a gene encoding protein responsible for RNA editing by site-specific deamination of adenosines. The enzyme can affect the gene expression by altering splice site recognition sequences and is associated with ADAR include Dyschromatosis Symmetrica Hereditaria and Aicardi-Goutieres Syndrome 6.

Characterization

The pegRNA in combination with ngRNA was used to test the efficiency Prime Editor 2. 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 on ADAR target site, proving the validity of the pegRNA design.

Fig. 1 Prime editing activity of PE2 at ADAR 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]