Arana Díaz, Álvaro JesúsVeiga Rúa, SaraCora Calvo, DiegoGonzález Gómez, Manuel AntonioSeijas Cerceda, AnaCarballeda Álvarez, MaialenPolo Montero, DavidCuesta, AlbertoPiñeiro Redondo, YolandaRivas Rey, JoséNovo, MercedesAl-Soufi, WajihMartínez Portela, PaulinoSánchez Piñón, LauraRobledo Sánchez, Diego2025-11-142025-11-142025Arana, Á. J., Veiga-Rua, S., Cora, D., Gónzalez-Gómez, M. A., Seijas, A., Carballeda, M., Polo, D., Cuesta, A., Piñeiro, Y., Rivas, J., Novo, M., Al-Soufi, W., Martínez, P., Sánchez, L., & Robledo, D. (2025). Comparative Analysis of CRISPR/Cas9 Delivery Methods in Marine Teleost Cell Lines. International Journal of Molecular Sciences, 26(21), 10703. https://doi.org/10.3390/ijms2621107031661-6596https://hdl.handle.net/10347/43780Gene editing technologies such as CRISPR/Cas9 have revolutionized functional genomics, yet their application in marine fish cell lines remains limited by inefficient delivery. This study compares three delivery strategies—electroporation, lipid nanoparticles (LNPs), and magnetofection using gelatin-coated superparamagnetic iron oxide nanoparticles (SPIONs)—for CRISPR/Cas9-mediated editing of the ifi27l2a gene in DLB-1 and SaB-1 cell lines. We evaluated transfection and editing efficiency, intracellular Cas9 localization, and genomic stability of the target locus. Electroporation achieved up to 95% editing in SaB-1 under optimized conditions, but only 30% in DLB-1, which exhibited locus-specific genomic rearrangements. Diversa LNPs enabled intracellular delivery and moderate editing (~25%) in DLB-1 but yielded only minimal editing in SaB-1, while SPION-based magnetofection resulted in efficient uptake but no detectable editing, highlighting post-entry barriers. Confocal imaging and fluorescence correlation spectroscopy suggested that nuclear localization and Cas9 aggregation may influence editing success, highlighting the importance of intracellular trafficking in CRISPR/Cas9 delivery. Our findings demonstrate that CRISPR/Cas9 delivery efficiency is cell line-dependent and governed by intracellular trafficking and genomic integrity. These insights provide a practical framework for optimizing gene editing in marine teleosts, advancing both basic research and selective breeding in aquacultureeng© 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)Attribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/CRISPRCas9Gene editingEditing efficiencyAquacultureSea bassSea breamjournal article10.3390/ijms2621107031422-0067open access