在非洲爪蟾胚胎中使用CRISPR/Cas9系统对参与神经嵴发育的基因进行高效基因组编辑。
Efficient genome editing of genes involved in neural crest development using the CRISPR/Cas9 system in Xenopus embryos.
作者信息
Liu Zhongzhen, Cheng Tina Tsz Kwan, Shi Zhaoying, Liu Ziran, Lei Yong, Wang Chengdong, Shi Weili, Chen Xiongfeng, Qi Xufeng, Cai Dongqing, Feng Bo, Deng Yi, Chen Yonglong, Zhao Hui
机构信息
Key Laboratory for Regenerative Medicine, Ministry of Education, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, SAR, China ; KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research of Common Diseases, Hong Kong, SAR, China.
Key Laboratory for Regenerative Medicine, Ministry of Education, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, SAR, China.
出版信息
Cell Biosci. 2016 Mar 31;6:22. doi: 10.1186/s13578-016-0088-4. eCollection 2016.
BACKGROUND
The RNA guided CRISPR/Cas9 nucleases have been proven to be effective for gene disruption in various animal models including Xenopus tropicalis. The neural crest (NC) is a transient cell population during embryonic development and contributes to a large variety of tissues. Currently, loss-of-function studies on NC development in X. tropicalis are largely based on morpholino antisense oligonucleotide. It is worthwhile establishing targeted gene knockout X. tropicails line using CRISPR/Cas9 system to study NC development.
METHODS
We utilized CRISPR/Cas9 to disrupt genes that are involved in NC formation in X. tropicalis embryos. A single sgRNA and Cas9 mRNA synthesized in vitro, were co-injected into X. tropicalis embryos at one-cell stage to induce single gene disruption. We also induced duplex mutations, large segmental deletions and inversions in X. tropicalis by injecting Cas9 and a pair of sgRNAs. The specificity of CRISPR/Cas9 was assessed in X. tropicalis embryos and the Cas9 nickase was used to reduce the off-target cleavages. Finally, we crossed the G0 mosaic frogs with targeted mutations to wild type frogs and obtained the germline transmission.
RESULTS
Total 16 target sites in 15 genes were targeted by CRISPR/Cas9 and resulted in successful indel mutations at 14 loci with disruption efficiencies in a range from 9.3 to 57.8 %. Furthermore, we demonstrated the feasibility of generation of duplex mutations, large segmental deletions and inversions by using Cas9 and a pair of sgRNAs. We observed that CRISPR/Cas9 displays obvious off-target effects at some loci in X. tropicalis embryos. Such off-target cleavages was reduced by using the D10A Cas9 nickase. Finally, the Cas9 induced indel mutations were efficiently passed to G1 offspring.
CONCLUSION
Our study proved that CRISPR/Cas9 could mediate targeted gene mutation in X. tropicalis with high efficiency. This study expands the application of CRISPR/Cas9 platform in X. tropicalis and set a basis for studying NC development using genetic approach.
背景
RNA 引导的 CRISPR/Cas9 核酸酶已被证明在包括热带爪蟾在内的各种动物模型中对基因破坏有效。神经嵴（NC）是胚胎发育过程中的一个短暂细胞群体，可分化为多种组织。目前，热带爪蟾中关于 NC 发育的功能丧失研究主要基于吗啉代反义寡核苷酸。利用 CRISPR/Cas9 系统建立靶向基因敲除的热带爪蟾品系来研究 NC 发育是很有必要的。
方法
我们利用 CRISPR/Cas9 破坏热带爪蟾胚胎中参与 NC 形成的基因。将体外合成的单个 sgRNA 和 Cas9 mRNA 在单细胞期共同注射到热带爪蟾胚胎中以诱导单基因破坏。我们还通过注射 Cas9 和一对 sgRNAs 在热带爪蟾中诱导双链突变、大片段缺失和倒位。在热带爪蟾胚胎中评估了 CRISPR/Cas9 的特异性，并使用 Cas9 切口酶来减少脱靶切割。最后，我们将具有靶向突变的 G0 嵌合蛙与野生型蛙杂交并获得了种系传递。
结果
CRISPR/Cas9 靶向了 15 个基因中的总共 16 个靶位点，并在 14 个位点成功产生了插入缺失突变，破坏效率在 9.3% 至 57.8% 之间。此外，我们证明了使用 Cas9 和一对 sgRNAs 产生双链突变、大片段缺失和倒位的可行性。我们观察到 CRISPR/Cas9 在热带爪蟾胚胎的某些位点显示出明显的脱靶效应。使用 D10A Cas9 切口酶可减少这种脱靶切割。最后，Cas9 诱导的插入缺失突变有效地传递给了 G1 后代。
结论
我们的研究证明 CRISPR/Cas9 可以高效介导热带爪蟾中的靶向基因突变。本研究扩展了 CRISPR/Cas9 平台在热带爪蟾中的应用，并为利用遗传方法研究 NC 发育奠定了基础。
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