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<p>The embryo survival rate is a critical concern in the genome editing for marine fish. The survival rates of genome-edited embryos of olive flounder and turbot injected using different mRNA purification methods were analyzed. Furthermore, RNA sequencing (RNA-seq) was used to investigate the impact of purification methods on embryo survival in the two flatfish species. Specifically, two RNA purification methods, Clean-up Column purification and LiCl/ethanol precipitation, were compared to analyze their effects on the genome-edited embryos of these two flatfish species. A 1–2-nL solution of 400-ng/µL gRNA was microinjected into the fertilized eggs of both olive flounder and turbot. The results indicate that, compared to Clean-up Column purification, gRNA purified using LiCl/ethanol precipitation resulted in a 4.73% decrease in hatching rate and a 19.61% higher deformity rate in olive flounder. In turbot, the proportion of viable embryos in the mid-gastrula stage decreased by 15.78%. Further RNA-seq revealed that microinjection and LiCl/ethanol-precipitated RNA affected embryonic development through their impact on cellular senescence, necroptosis and apoptosis, embryogenesis, and osmotic regulation in both species. This study provided clues on how to increase the survival rates of flatfish and other marine fish species during genome editing.</p>

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Effects of RNA purification methods on genome editing for olive flounder and turbot

  • 期刊论文

  • 发表日期：

    2026年6月29日

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  • Sishi Qin,
  • Lijuan Wang,
  • Zhihao Wu,
  • Yaobo Jin,
  • Yansong Liu,
  • Feng You,
  • Xungang Tan

摘要

The embryo survival rate is a critical concern in the genome editing for marine fish. The survival rates of genome-edited embryos of olive flounder and turbot injected using different mRNA purification methods were analyzed. Furthermore, RNA sequencing (RNA-seq) was used to investigate the impact of purification methods on embryo survival in the two flatfish species. Specifically, two RNA purification methods, Clean-up Column purification and LiCl/ethanol precipitation, were compared to analyze their effects on the genome-edited embryos of these two flatfish species. A 1–2-nL solution of 400-ng/µL gRNA was microinjected into the fertilized eggs of both olive flounder and turbot. The results indicate that, compared to Clean-up Column purification, gRNA purified using LiCl/ethanol precipitation resulted in a 4.73% decrease in hatching rate and a 19.61% higher deformity rate in olive flounder. In turbot, the proportion of viable embryos in the mid-gastrula stage decreased by 15.78%. Further RNA-seq revealed that microinjection and LiCl/ethanol-precipitated RNA affected embryonic development through their impact on cellular senescence, necroptosis and apoptosis, embryogenesis, and osmotic regulation in both species. This study provided clues on how to increase the survival rates of flatfish and other marine fish species during genome editing.