Figure2 . The constructed pegRNA mediates the transversion of
mutated base in leptin receptor of db/db mice. (A) Targeting strategy
with pegRNA system, gRNA sequence (orange), RT template (blue),
nucleotide to be correcte (red), primer binding site (green), and pegRNA
stem (purple). (B) GFP microscope images after FDB electroporation of
constructed pegRNAs. (C) The upper panel shows the sanger sequencing of
leptin receptor from db/db mice. The red arrows represent the mutated
sequence. The lower panel shows the transversion of T to G. The K is the
symbol of Guanine and Thymine. The red arrow means mixing of two
nucleotides.
3.3. Leptin Signaling Activity is Enhanced after Prime Editing of Leptin
Receptor in the Flexor Digitorum Brevis Muscle
We next to investigate whether leptin receptor protein function was also
rescued after prime editing treatment. As it is well known, STAT3 is the
downstream factor of leptin/leptin receptor signaling pathway[18]. Thus, we performed Western blot analysis to
probe STAT3 expression. Indeed,the results indicated a notable
upregulation in STAT3 expression within the FDB muscle treated with
prime editing as compared with untreated muscle samples. Furthermore, we
found that both pegRNA8 (P = 0.033) and pegRNA10 (P = 0.014) worked
equally well in rescuing STAT3 signaling (Figure 3). This observation
suggests that in vivo delivery of PE constructs effectively promoted
base transversion of mutant leptin receptor and enhanced downstream
STAT3 signaling pathway in db/db mice. Thus, prime editing could produce
functional leptin receptor in db/db skeletal muscles.