Using CRISPR/Cas9 to identify genes required for mechanosensory neuron development and function.
Using CRISPR/Cas9 to identify genes required for mechanosensory neuron development and function.
复制标题
使用 CRISPR/Cas9 识别机械感觉神经元发育和功能所需的基因。
DOI：
10.1101/2023.05.08.539861
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
V
中科院分区：
文献类型：
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作者：
Johnson,ChristopherJ;Kulkarni,Akhil;Buxton,WilliamJ;Hui,TszY;Kayastha,Anusha;Khoja,AlwinA;Leandre,Joviane;Mehta,VanshikaV;Ostrowski,Logan;Pareizs,EricaG;Scotto,RebeccaL;Vargas,Vanesa;Vellingiri,RaveenaM;Verzino,Giulia;V
被囊动物是海洋非脊椎动物的脊索动物，包括脊椎动物的姐妹群。大多数被囊动物有一个双相的生命周期，在游泳的幼虫和无柄的成虫之间交替。最近的进展揭示了被囊动物幼虫的能力，感觉到一个适当的解决和启动变态基板的神经基础。在高度易处理的实验室模型中，被囊玻璃海鞘粗壮的工作表明，感觉神经元嵌入在前乳头的mechanosensory刺激，以触发幼虫的尾部收缩，并启动变态的过程。在这里，我们利用Ciona的低成本和简单性，通过使用组织特异性CRISPR/Cas9介导的诱变来筛选可能参与机械感觉和变态的基因，在本科“顶点”研究课程的背景下。这个小屏幕显示了至少一个基因，Vamp 1/2/3，这似乎对乳头触发变态的能力至关重要。我们还提供与本课程相关的分步协议和教程，希望它可以在类似的基于CRISPR的实验室课程中复制，只要Ciona可用。
Tunicates are marine, non-vertebrate chordates that comprise the sister group to the vertebrates. Most tunicates have a biphasic lifecycle that alternates between a swimming larva and a sessile adult. Recent advances have shed light on the neural basis for the tunicate larva's ability to sense a proper substrate for settlement and initiate metamorphosis. Work in the highly tractable laboratory model tunicate Ciona robusta suggests that sensory neurons embedded in the anterior papillae transduce mechanosensory stimuli to trigger larval tail retraction and initiate the process of metamorphosis. Here, we take advantage of the low-cost and simplicity of Ciona by using tissue-specific CRISPR/Cas9-mediated mutagenesis to screen for genes potentially involved in mechanosensation and metamorphosis, in the context of an undergraduate ‘capstone’research course. This small screen revealed at least one gene, Vamp1/2/3, which appears crucial for the ability of the papillae to trigger metamorphosis. We also provide step-by-step protocols and tutorials associated with this course, in the hope that it might be replicated in similar CRISPR-based laboratory courses wherever Ciona are available.