Highly Efficient Knockout of a Squid Pigmentation Gene
Highly Efficient Knockout of a Squid Pigmentation Gene
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DOI：
10.1016/j.cub.2020.06.099
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发表时间：
2020-09-07
期刊：
影响因子：
9.2
通讯作者：
Rosenthal, Joshua J. C.
中科院分区：
文献类型：
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作者：
Crawford, Karen;Quiroz, Juan F. Diaz;Rosenthal, Joshua J. C.
以鱿鱼为模型的精液研究导致了神经生物学的突破。例如，鱿鱼的巨大轴突和突触为我们目前对动作电位[1]、细胞间离子梯度[2]、电压依赖性离子通道[3]、分子马达[4-7]和突触传递[8-11]的理解奠定了基础。尽管它们在解剖学上有优势，但在过去的几十年里，随着研究人员转向遗传学上易于处理的系统，鱿鱼作为模型的使用逐渐减少。然而，最近有两项关键进展使开发鱿鱼遗传操纵技术成为可能。第一个是用于靶向基因破坏的CRISPR-Cas9系统，这是一种主要的物种不可知方法[12，13]。第二个是几个头足类物种的基因组测序[14-16]。如果在基因上变得易于控制，鱿鱼和其他头足类动物提供了丰富的生物学新奇，可能会刺激发现。在无脊椎动物中，它们不仅拥有迄今为止最大的大脑，它们还表达了最复杂的行为[17]。在本文中，我们证明了使用CRISPR-Cas9在鱿鱼Doryteuthis pealeii中的有效基因敲除。Ommochromes是鱿鱼视网膜和色素细胞中发现的色素，是色氨酸的衍生物，其合成的第一个关键步骤通常由色氨酸2，3双加氧酶（TDO [18-20]）催化。敲除鱿鱼胚胎中的TDO有效地消除了色素沉着。通过在早期发育期间精确定时CRISPR-Cas9的递送，可以精细地控制色素沉着的程度。基因分型显示敲除效率通常大于90%。这项研究代表了一个关键的进步，使鱿鱼遗传温顺。
Seminal studies using squid as a model led to breakthroughs in neurobiology. The squid giant axon and synapse, for example, laid the foundation for our current understanding of the action potential [1], ionic gradients across cells [2], voltage-dependent ion channels [3], molecular motors [4-7], and synaptic transmission [8-11]. Despite their anatomical advantages, the use of squid as a model receded over the past several decades as investigators turned to genetically tractable systems. Recently, however, two key advances have made it possible to develop techniques for the genetic manipulation of squid. The first is the CRISPR-Cas9 system for targeted gene disruption, a largely species-agnostic method [12, 13]. The second is the sequencing of genomes for several cephalopod species [14-16]. If made genetically tractable, squid and other cephalopods offer a wealth of biological novelties that could spur discovery. Within invertebrates, not only do they possess by far the largest brains, they also express the most sophisticated behaviors [17]. In this paper, we demonstrate efficient gene knockout in the squid Doryteuthis pealeii using CRISPR-Cas9. Ommochromes, the pigments found in squid retinas and chromatophores, are derivatives of tryptophan, and the first committed step in their synthesis is normally catalyzed by Tryptophan 2,3 Dioxygenase (TDO [18-20]). Knocking out TDO in squid embryos efficiently eliminated pigmentation. By precisely timing CRISPR-Cas9 delivery during early development, the degree of pigmentation could be finely controlled. Genotyping revealed knockout efficiencies routinely greater than 90%. This study represents a critical advancement toward making squid genetically tractable.