Abstract
Albinism is the most common color variation described in fish and is a fascinating trait of some ornamental fish species. Albino mutants can be generated by knocking out core genes affecting melanin synthesis like slc45a2 in several fish species. However, genetic mutation remains challenging for species with unknown genome information. In this study, we generated albino mutants in two selected ornamental fish species, royal farlowella (Sturisoma panamense), and redhead cichlid (Vieja melanura). For this purpose, we carried out phylogenetic analyses of fish slc45a2 sequences and identified a highly conserved region among different fish species. A pair of degenerate primers spanning this region was designed and used to amplify a conserved slc45a2 fragment of 340 bp from the two fish species. Based on the amplified sequences, a target site in the 6th exon was used for designing guide RNA and this targeted site was first verified by the CRISPR/Cas9 system in the zebrafish (Danio rerio) model for the effectiveness. Then, specific guide RNAs were designed for the two ornamental fish species and tested. Most of the injected larvae completely lost black pigment over the whole body and eyes. DNA sequencing confirmed a high degree of mutation at the targeted site. Overall, we described a fast and efficient method to generate albino phenotype in fish species by targeting the conserved 6th exon of slc45a2 gene for genome editing via CRISPR/Cas9 and this approach could be a new genetic tool to generate desirable albino ornamental fish.
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Data Availability
The datasets used in the present study are available from the corresponding author on reasonable request.
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Acknowledgements
ZR is supported by the NUS industry-relevant Ph.D scholarship. We thank Ms. Siangna Tan and Qian Hu staff to maintain fish aquaria.
Funding
This work was financially supported by a collaboration fund with Qian Hu Fish Farm of Singapore (R154000B27592) and Shandong Provincial Natural Science Foundation (ZR2021QH300).
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Changqing Zhang: conceptualization, methodology, investigation, data curation, formal analysis, supervision, and writing—original draft. Ziheng Ren: conceptualization, methodology, investigation, data curation, and writing—editing. Zhiyuan Gong: conceptualization, methodology, funding acquisition, resources, supervision, project administration, and writing—review and editing.
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Zhang, C., Ren, Z. & Gong, Z. Generation of Albino Phenotype in Ornamental Fish by CRISPR/Cas9-Mediated Genome Editing of slc45a2 Gene. Mar Biotechnol 25, 281–290 (2023). https://doi.org/10.1007/s10126-023-10204-9
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DOI: https://doi.org/10.1007/s10126-023-10204-9