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.