Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish
Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish
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DOI：
10.3791/60253
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发表时间：
2019-10-01
影响因子：
1.2
通讯作者：
Gallant, Jason R.
中科院分区：
文献类型：
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作者：
Constantinou, Savvas J.;Linh Nguyen;Gallant, Jason R.
电接受和电发生在脊椎动物的进化史上发生了变化。这些独立衍生的表型具有惊人的趋同程度，它们具有共同的遗传结构。裸子鱼和硬骨鱼这两种种类丰富的硬骨鱼分支能够产生和探测弱电场，因此被称为弱电鱼，它们的许多趋同特征或许就是最好的例证。自从发现弱电鱼利用电来感知周围环境和交流以来的50年里，越来越多的科学家在发育进化、系统和电路神经科学、细胞生理学、生态学、进化生物学和行为学方面获得了巨大的见解。最近，电鱼的基因组资源激增。这些资源的使用已经促进了对这些物种的基因型和表型之间联系的重要见解。整合基因组学数据与弱电鱼表型数据的主要障碍是目前缺乏功能基因组学工具。我们在此报告了在弱电鱼中利用内源性DNA修复机制进行CRISPR/Cas9诱变的完整方案。我们使用CRISPR/Cas9靶向钠通道基因scn4aa第一外显子的插入子和点突变，证明该方案在mormyrid物种Brienomyrus brachyistius和裸子形Brachyhypopomus gauderio中同样有效。使用该方案，从两个物种获得胚胎并进行基因分型，以确认在钠通道scn4aa的第一外显子中存在预测的突变。与未注射大小匹配的对照相比，记录显示电器官放电幅度降低，证实了敲除成功表型。
Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.