Cas9 ribonucleoprotein complex allows direct and rapid analysis of coding and noncoding regions of target genes in Pleurodeles waltl development and regeneration.
Cas9 ribonucleoprotein complex allows direct and rapid analysis of coding and noncoding regions of target genes in Pleurodeles waltl development and regeneration.
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DOI：
10.1016/j.ydbio.2018.09.008
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发表时间：
2018-11
影响因子：
2.7
通讯作者：
Miyuki Suzuki;Toshinori Hayashi;Takeshi Inoue;K. Agata;M. Hirayama;Miyuzu Suzuki;S. Shigenobu;T. Takeuchi;Takashi Yamamoto;Ken-ichi T. Suzuki
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文献类型：
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作者：
Miyuki Suzuki;Toshinori Hayashi;Takeshi Inoue;K. Agata;M. Hirayama;Miyuzu Suzuki;S. Shigenobu;T. Takeuchi;Takashi Yamamoto;Ken-ichi T. Suzuki
蝾螈具有非凡的再生器官的能力，几个世纪以来一直被用于研究。然而，繁重的育种工作阻碍了纽特的反向遗传学策略。在这里，我们提出了一个简单而有效的基因敲除方法，使用Cas9核糖核蛋白复合体(RNP)在侧耳中进行基因敲除，这是一个适合使用反向遗传学进行再生生物学研究的物种。大多数创建者对每个目标基因(酪氨酸酶、Pax6、tbx5)表现出严重的表型；值得注意的是，所有酪氨酸酶Cas9 RNP注射的胚胎显示完全白化。此外，对注射Cas9 RNP的胚胎进行的扩增序列分析显示，创始人的目标基因座几乎完全破坏了双等位基因，从而可以在F0代进行直接的表型分析。此外，我们在一年内演示了酪氨酸作为完整的F1后代的世代。最后，我们将这种方法扩展到非编码调控元件的分析，通过靶向Sonic Hedgehog的肢体特异性增强子，称为极化活动调控序列区(ZRS；也称为MFCS1)。ZRS的破坏导致了肢体再生中的手指畸形。从这些结果中，我们相信这种高效的基因敲除方法将加速后基因组时代的基因功能分析。
Newts have remarkable ability to regenerate their organs and have been used in research for centuries. However, the laborious work of breeding has hampered reverse genetics strategies in newt. Here, we present simple and efficient gene knockout using Cas9 ribonucleoprotein complex (RNP) inPleurodeles waltl, a species suitable for regenerative biology studies using reverse genetics. Most of the founders exhibited severe phenotypes against each target gene (tyrosinase,pax6,tbx5); notably, alltyrosinaseCas9 RNP-injected embryos showed complete albinism. Moreover, amplicon sequencing analysis of Cas9 RNP-injected embryos revealed virtually complete biallelic disruption at target loci in founders, allowing direct phenotype analysis in the F0generation. In addition, we demonstrated the generation oftyrosinasenull F1offspring within a year. Finally, we expanded this approach to the analysis of noncoding regulatory elements by targeting limb-specific enhancer of sonic hedgehog, known as the zone of polarizing activity regulatory sequence (ZRS; also called MFCS1). Disruption of ZRS led to digit deformation in limb regeneration. From these results, we are confident that this highly efficient gene knockout method will accelerate gene functional analysis in the post-genome era of salamanders.