CRISPR/Cas9-Mediated Disruption of Endo16 Cis-Regulatory Elements in Sea Urchin Embryos
CRISPR/Cas9-Mediated Disruption of Endo16 Cis-Regulatory Elements in Sea Urchin Embryos
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DOI：
10.3390/fishes8020118
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发表时间：
2023-02
期刊：
影响因子：
2.3
通讯作者：
Lili Xing;Lingyu Wang;Femke Roos;Michelle Lee;G. Wray
中科院分区：
文献类型：
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作者：
Lili Xing;Lingyu Wang;Femke Roos;Michelle Lee;G. Wray
海胆已成为全球重要的海水养殖物种，也是发育生物学中的无脊椎模式生物。顺式调节元件（增强子）通过调节基因表达控制发育和生理。影响这些序列功能的突变可能有助于表型多样性。顺式调控靶点为未来提供了新的育种潜力。鉴于对Endo 16调控区的深入研究，本文利用CRISPR/Cas9系统破坏Endo 16在杂色绿绒螯蟹胚胎发育中的增强子。我们针对Endo 16模块A（调控序列的最近端区域，其特别激活植物板和原肠中的转录）设计了六种gRNA，并发现Endo 16模块A破坏的胚胎在受精后20小时未能经历原肠胚形成。该结果部分地表型模仿Endo 16的吗啉代敲除。此外，我们进行了qPCR和克隆测序实验来验证这些结果。虽然没有发现突变定期从测序受影响的个人，我们讨论了一些潜在的原因。本研究为研究海胆顺式调控元件的功能提供了一种可行的方法，为棘皮动物精密育种技术创新和水产养殖业发展提供了参考。
Sea urchins have become significant mariculture species globally, and also serve as invertebrate model organisms in developmental biology. Cis-regulatory elements (enhancers) control development and physiology by regulating gene expression. Mutations that affect the function of these sequences may contribute to phenotypic diversity. Cis-regulatory targets offer new breeding potential for the future. Here, we use the CRISPR/Cas9 system to disrupt an enhancer of Endo16 in developing Lytechinus variegatus embryos, in consideration of the thorough research on Endo16’s regulatory region. We designed six gRNAs against Endo16 Module A (the most proximal region of regulatory sequences, which activates transcription in the vegetal plate and archenteron, specifically) and discovered that Endo16 Module A-disrupted embryos failed to undergo gastrulation at 20 h post fertilization. This result partly phenocopies morpholino knockdowns of Endo16. Moreover, we conducted qPCR and clone sequencing experiments to verify these results. Although mutations were not found regularly from sequencing affected individuals, we discuss some potential causes. In conclusion, our study provides a feasible and informative method for studying the function of cis-regulatory elements in sea urchins, and contributes to echinoderm precision breeding technology innovation and aquaculture industry development.