Targeted gene disruption by use of CRISPR/Cas9 ribonucleoprotein complexes in the water flea Daphnia pulex
Targeted gene disruption by use of CRISPR/Cas9 ribonucleoprotein complexes in the water flea Daphnia pulex
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DOI：
10.1111/gtc.12589
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发表时间：
2018-06-01
期刊：
影响因子：
2.1
通讯作者：
Iguchi, Taisen
中科院分区：
文献类型：
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作者：
Hiruta, Chizue;Kakui, Keiichi;Iguchi, Taisen
微甲壳类动物水蚤（Daphnia pulex）的适应性生活史显示出对环境变化的可塑性，是环境、生态、进化和发育基因组学研究的重要模型。尽管该物种的全基因组序列和组学数据已经积累，但迄今为止，用于分析基因功能的可用工具仅限于RNAi （RNA干扰）和TALEN（转录激活因子样效应核酸酶）系统。因此，CRISPR/Cas9（聚集规律间隔的短回文重复序列/CRISPR相关9）系统的发展有望进一步提高D.pulex的遗传可追溯性，并促进对该物种的了解。在这项研究中，我们利用CRISPR/Cas9核糖核蛋白复合物（Cas9 RNPs）开发了一种D.pulex基因组编辑系统。我们首先组装了一种针对远端缺失基因（Dll）的CRISPR单导RNA (sgRNA)，该基因编码一种同源结构域转录因子，对无脊椎动物和脊椎动物的远端肢体发育至关重要。然后，我们将Cas9 RNPs注射到卵子中，并通过T7内切酶I检测评估其在体内的活性。注射后的胚胎显示第二天线形成缺陷，附属物发育紊乱，Dll位点检测到indel突变，表明该技术成功敲除了靶基因。
The microcrustacean Daphnia pulex is an important model for environmental, ecological, evolutionary and developmental genomics because its adaptive life history displays plasticity in response to environmental changes. Even though the whole-genome sequence is available and omics data have actively accumulated for this species, the available tools for analyzing gene function have thus far been limited to RNAi (RNA interference) and TALEN (the transcription activator-like effector nuclease) systems. The development of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) system is thus expected to further increase the genetic tractability of D.pulex and to advance the understanding of this species. In this study, we developed a genome editing system for D.pulex using CRISPR/Cas9 ribonucleoprotein complexes (Cas9 RNPs). We first assembled a CRISPR single-guide RNA (sgRNA) specific to the Distal-less gene (Dll), which encodes a homeodomain transcription factor essential for distal limb development in invertebrates and vertebrates. Then, we injected Cas9 RNPs into eggs and evaluated its activity in vivo by a T7 endonuclease I assay. Injected embryos showed defective formation of the second antenna and disordered development of appendages, and indel mutations were detected in Dll loci, indicating that this technique successfully knocked out the target gene.