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CRISPR/Cas9-mediated knock-in of masu salmon (Oncorhyncus masou) elongase gene in the melanocortin-4 (mc4r) coding region of channel catfish (Ictalurus punctatus) genome

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Abstract

Channel catfish, Ictalurus punctatus, have limited ability to synthesize Ω-3 fatty acids. The ccβA-msElovl2 transgene containing masu salmon, Oncorhynchus masou, elongase gene driven by the common carp, Cyprinus carpio, β-actin promoter was inserted into the channel catfish melanocortin-4 receptor (mc4r) gene site using the two-hit two-oligo with plasmid (2H2OP) method. The best performing sgRNA resulted in a knockout mutation rate of 92%, a knock-in rate of 54% and a simultaneous knockout/knock-in rate of 49%. Fish containing both the ccβA-msElovl2 transgene knock-in and mc4r knockout (Elovl2) were 41.8% larger than controls at 6 months post-hatch (p = 0.005). Mean eicosapentaenoic acid (EPA, C20:5n-3) levels in Elov2 mutants and mc4r knockout mutants (MC4R) were 121.6% and 94.1% higher than in controls, respectively (p = 0.045; p = 0.025). Observed mean docosahexaenoic acid (DHA, C22:6n-3) and total EPA + DHA content was 32.8% and 45.1% higher, respectively, in Elovl2 transgenic channel catfish than controls (p = 0.368; p = 0.025). To our knowledge this is the first example of genome engineering to simultaneously target transgenesis and knock-out a gene in a commercially important aquaculture species for multiple improved performance traits. With a high transgene integration rate, improved growth, and higher omega-3 fatty acid content, the use of Elovl2 transgenic channel catfish appears beneficial for application on commercial farms.

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All data generated or analyzed during this study are included in this published article and its supplemental files.

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Acknowledgements

The authors thank Professor Sushil Adhikari’s research group and the Center for Bioenergy and Bioprodroducts at Auburn University and Professor D. Allen Davis’ research group at Auburn University for assisting with the fatty acid composition. This project was partially funded by USDA-NIFA award 2015-67015-23488 to Roger Cone and by an Ocean University China-Auburn University grant to Rex Dunham, Zhenkui Qin, and Baofeng Su.

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  1. School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA

    Michael Coogan, De Xing, Baofeng Su, Veronica Alston, Andrew Johnson, Mohd Khan, Karim Khalil, Ahmed Elaswad, Shangjia Li, Jinhai Wang, Cuiyu Lu, Wenwen Wang, Darshika Hettiarachchi, Mei Shang, Tasnuba Hasin, Zhenkui Qin, Ian A. E. Butts & Rex A. Dunham

  2. Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh, 2202, Bangladesh

    Mohd Khan

  3. Life Sciences Institute, University of Michigan, Ann Arbor, MI, 48109, USA

    Roger Cone

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Contributions

MC conceived and designed the study, collected and analyzed data, generated and cultured the fish, and wrote the manuscript. DX developed the ccβA-msElovl2 plasmid containing the masu salmon, Onchorhynchus masou, elongase gene driven by the common carp β-actin promoter and assisted in fatty acid analysis. BS was a supervisory team leader, developed the primers and analyzed the data. VA, AJ, JW, SL, WW, DH, and TH collected data. AJ, JW, SL, DX, and BS cultured fish. MK, AE and KK developed brood stock. CL, BS, MK, and MS developed laboratory methodology. ZQ and RC assisted with initial research stages of elongase research within our laboratory. Ian Butts was a supervisory team member and assisted with statistical analyses. RD was the principal investigator and assisted with writing. All authors have read and agreed to the published version of the manuscript.

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Coogan, M., Xing, D., Su, B. et al. CRISPR/Cas9-mediated knock-in of masu salmon (Oncorhyncus masou) elongase gene in the melanocortin-4 (mc4r) coding region of channel catfish (Ictalurus punctatus) genome. Transgenic Res 32, 251–264 (2023). https://doi.org/10.1007/s11248-023-00346-w

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