Figure 1. Selection of gRNA targeting the mutated leptin receptor in db/db mice. The analysis of Sanger sequencing of endonuclease activity of each gRNA/Cas9 complex to induce double-strand breakage at genomic target site.
3.2. Prime Editing of Leptin Receptor Results in Nucleotide Transversion from T to G.
Next, we generated two kinds of pegRNA8 and pegRNA10 based on gRNA3 which has different length of prime binding sites (Figure 2A). Then, we addressed whether pegRNA was able to edit the nucleotide in Leptin receptor of db/db mice. Using the FDB electroporation method to deliver our pegRNAs construct, we were able to isolate GFP positive myofibers (Figure 2B), subsequently amplified the PCR product for Sanger sequencing. The db/db mouse’s mutation is caused by G to T. When treated with our pegRNA, we noted the T was transversion with G, denoting a mix of two nucleotides (i.e G and T) (Figure 2C). This result suggested that the PE constructs could correct leptin receptor point mutation in vivo.
A.