RT Journal Article
T1 Comparative Analysis of CRISPR/Cas9 Delivery Methods in Marine Teleost Cell Lines
A1 Arana Díaz, Álvaro Jesús
A1 Veiga Rúa, Sara
A1 Cora Calvo, Diego
A1 González Gómez, Manuel Antonio
A1 Seijas Cerceda, Ana
A1 Carballeda Álvarez, Maialen
A1 Polo Montero, David
A1 Cuesta, Alberto
A1 Piñeiro Redondo, Yolanda
A1 Rivas Rey, José
A1 Novo, Mercedes
A1 Al-Soufi, Wajih
A1 Martínez Portela, Paulino
A1 Sánchez Piñón, Laura
A1 Robledo Sánchez, Diego
K1 CRISPR
K1 Cas9
K1 Gene editing
K1 Editing efficiency
K1 Aquaculture
K1 Sea bass
K1 Sea bream
AB Gene 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 aquaculture
PB MDPI
SN 1661-6596
YR 2025
FD 2025
LK https://hdl.handle.net/10347/43780
UL https://hdl.handle.net/10347/43780
LA eng
NO Arana, 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/ijms262110703
NO This research was funded by the collaborative project at Campus Terra, University of Santiago de Compostela, within the framework of the Collaboration Agreement between the USC and the Department of Culture, Education, Vocational Training, and Universities, and it was also funded by Fundación Caixa Rural Galega Tomás Notario Vacas within the project Optimizing CRISPR/Cas9 genome editing to improve disease resistance in aquaculture. Additionally, this research was carried out under the framework of Spain’s Recovery and Resilience Plan, specifically under investment line nº 1 and component number 17, which includes the Complementary RTDI Plan for Marine Science. This work also was funded by the European Union ERC Starting Grant programme 2022 under grant agreement No 101076432 (FishTRIM). DR was supported by the Oportunius programme of the Axencia Galega de Innovación (GAIN, Xunta de Galicia), and by BBSRC Institute Strategic Grants to the Roslin Institute (BBS/E/20002172, BBS/E/D/30002275, BBS/E/D/10002070 and BBS/E/RL/230002A). D.P. was supported by a Ramón y Cajal grant (RYC2023-044793-I) funded by the Ministerio de Ciencia, Innovación y Universidades (MICIU), Agencia Estatal de Investigación (AEI), and ESF+ (10.13039/501100011033). Finally, this work was supported by project PID2022-137821OB-C31, funded by the Ministerio de Ciencia e Innovación, 2022
DS Minerva
RD 22 abr 2026