Methodological Evaluation of a P2C-Based ReMOT CRISPR/Cas9 System in Aedes aegypti

Mosquito-borne infectious diseases remain a major challenge to public health, highlighting the need for efficient and accessible gene editing approaches. Receptor-mediated ovary transduction of cargo (ReMOT) offers an alternative to embryonic microinjection, in which P2C, an ovary-targeting peptide, enables ovarian delivery of the editing components. However, key design parameters and operational boundaries of the P2C-based ReMOT system have not been clearly defined. Here, we performed a methodological evaluation of the P2C-mediated ReMOT CRISPR/Cas9 system in Aedes aegypti. Cas9-P2C fusion proteins with different configurations were constructed and assessed through ovarian targeting assays, in vitro cleavage analyses, and in vivo gene editing experiments. Our results show that full-length Cas9-P2C fusion proteins exhibit nuclease activity and enable effective ovarian delivery. In contrast, linear truncation of the P2C peptide markedly reduced ovarian targeting, indicating a dependence on structural integrity. Using this delivery strategy, we generated kynurenine monooxygenase (KMO) edited mosquitoes, demonstrating feasibility under the conditions tested. In addition, protein injection was also associated with reduced reproductive performance, providing physiological reference for ReMOT applications. Overall, this study defines the key design parameters and operational boundaries of the P2C-based ReMOT system, providing methodological guidance for its application and optimization in future mosquito genetic studies.