Figure3 . Western blot analysis of STAT3 in flexor digitorum brevis (FDB) muscle after prime editing with constructed pegRNAs (pegRNA8 and pegRNA10), the ipsilateral extensor digitorum longus (EDL) muscle was used as untreated control (pegRNA8 vs Untreated control: P = 0.033; pegRNA10 vs Untreated control: P = 0.014; pegRNA8 vs pegRNA10: P = 0.811).
4. Discussion
In this study, we constructed pegRNA to target mutated leptin receptor in the db/db mice using FDB muscle electroporation, aiming to evaluate the in vivo efficiency of pegRNA-mediated genome editing. Our results demonstrated successful base transversion mediated by the constructed pegRNA. More importantly, corrected leptin receptor protein functioned normally as evidenced by downstream STAT3 signaling. These findings underscore the feasibility of using pegRNA as a valuable tool for targeted genome editing in vivo. It highlights the potential of PE as a promising therapeutic approach to manipulate point-mutations in a litany of diseases, providing new avenues for potential therapeutic interventions.
While in vivo rescue of the leptin receptor has been performed in db/db mice [19], we believe our study has several advantages over the conventional gene delivery approaches. Our study focused on ”correct” the point mutation in endogenous leptin receptor gene. Thus, we only mended endogenous leptin receptor gene without altering its regulatory elements, such as 3’UTR, 5’UTR, etc. Therefore, the leptin receptor gene expression pattern, expression level, and tissue distribution in prime editing treated db/db mice should be very similar as those in wild type animal, which is very challenging in conventional gene delivery approach. In addition, PE is a ’search-and-replace’ genome editing technology. Unlike CRISPR-Cas9, it does not require DBSs or donor DNA templates to achieve precise modifications of the target DNA sequence [6]. In contrast, base editors, although capable of introducing transition point mutations without DSBs, currently lack the ability to install transversion point mutations, precise insertions, or precise deletions[5]. Furthermore, it can inadvertently induce bystander mutations when multiple target nucleotides exist within the editing window, and limitations in PAM availability may hinder targeting certain bases [5]. However, PE does not require a precisely positioned PAM sequence, thus providing greater flexibility and precision in targeting specific genomic loci[6]. This targeting flexibility allows for a wide range of editing possibilities, enabling precise alterations with enhanced accuracy [6,17]. The success of prime editing has been evident in numerous studies conducted in diverse cell types and animal models, underscoring its potential for therapeutic applications [20-22].
The translation of prime editing into a therapeutic approach in vivo requires careful consideration and strategic planning. While the efficiency and precision of prime editing have been demonstrated in experimental settings, the delivery of prime editing components into target cells or tissues remains a significant challenge. Various methods involving viral vectors, nanoparticles, or direct injection have been explored to ensure delivery [10,23-28]. To further advance this therapeutic approach, our ongoing efforts are dedicated to exploring efficient delivery strategies for the constructed pegRNAs into target cells in vivo and ensuring a desired therapeutic outcome.
5. Conclusions
In conclusion, our present study underscores the efficacy and precision of prime editing as a potent method for the correction of genetic mutations within the genome.
Author Contributions: Conceptualization, K.E.L., Y.X., D.L., H.Z.; methodology, K.E.L., Y.X. and B.G.; formal analysis, Y.X..; investigation, K.E.L., Y.X., B.G., J.K., N.K., M.H., Z.Z.; data curation, Y.X., Z.Z., D.G., and H.Z.; writing—original draft, K.E.L., Y.X., Z.Z., D.G., and H.Z.; writing—review and editing, all authors; supervision, H.Z.; funding acquisition, H.Z.. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by National Institutes of Health (NIH) (Grant number: EY032583; HL153876; EY030621; AR067766) and American Heart Association (AHA) (Grant number: 23TPA1142638).
Institutional Review Board Statement: All murine experiments in this study were performed following The Ohio State University Institutional Animal Care and Use Committee (IACUC) approved protocols (#2016A00000017).
Data Availability Statement: The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.
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