Sea urchins are used as a model organism for research on developmental biology and gene regulatory networks during early development. Gene knockdown by microinjection of morpholino antisense oligonucleotide (MASO) has been used to analyze gene function in early sea urchin embryos. However, as the effect of MASO is not long lasting, it is impossible to perturb genes expressed during late development by MASO. Recent advances in genome editing technologies have enabled gene modification in various organisms. We previously reported genome editing in the sea urchin Hemicentrotus pulcherrimus using zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN); however, the efficiencies of these technologies were not satisfactory. Here, we applied clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated nuclease 9 (Cas9) technology to knock out the Pks1 gene in H. pulcherrimus. When sgRNAs targeting Pks1, which is required for the biosynthesis of larval pigment, were microinjected into fertilized eggs with SpCas9 mRNA, high-efficiency mutagenesis was achieved within 24 hr post fertilization and SpCas9/sgRNA-injected pluteus larvae had an albino phenotype. One of the sgRNAs yielded 100% mutagenesis efficiency, and no off-target effect was detected. In addition, the albino phenotype was maintained in juvenile sea urchins after metamorphosis, and the knockout sea urchins survived for at least one year and grew to albino adult sea urchins. These findings suggest that knockout adult sea urchins were successfully established and the CRISPR-Cas9 system is a feasible method for analyzing gene functions from late developmental to adult stage.

中文翻译：

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通过使用CRISPR-Cas9系统建立敲除的成年海胆。

海胆被用作模型生物，用于在早期发育过程中研究发育生物学和基因调控网络。通过微注射吗啉代反义寡核苷酸（MASO）进行基因敲除已用于分析海胆早期胚胎的基因功能。但是，由于MASO的作用不会持续太久，因此不可能干扰MASO在后期发育过程中表达的基因。基因组编辑技术的最新进展已实现了各种生物体的基因修饰。我们之前曾报道过使用锌指核酸酶（ZFN）和转录激活因子样效应核酸酶（TALEN）在海胆Hemicentrotus pulcherrimus中进行基因组编辑；但是，这些技术的效率并不令人满意。这里，我们应用了聚类的规则间隔的短回文重复序列（CRISPR）-CRISPR相关的核酸酶9（Cas9）技术来敲除p.1基因在H. pulcherrimus中。当将具有幼虫色素生物合成所需的靶向Pks1的sgRNA微注射到带有SpCas9 mRNA的受精卵中时，在受精后24小时内实现了高效诱变，并且注射了SpCas9 / sgRNA的夜蛾幼虫具有白化病表型。一种sgRNA产生100％的诱变效率，未检测到脱靶效应。此外，变态后的少年海胆中还保留有白化病表型，基因敲除的海胆存活了至少一年并长成了白化病成年海胆。