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.