Rapid and efficient analysis of gene function using CRISPR-Cas9 in Xenopus tropicalis founders
Rapid and efficient analysis of gene function using CRISPR-Cas9 in Xenopus tropicalis founders
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DOI：
10.1111/gtc.12379
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发表时间：
2016-07-01
期刊：
影响因子：
2.1
通讯作者：
Suzuki, Ken-ichi T.
中科院分区：
文献类型：
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作者：
Shigeta, Mitsuki;Sakane, Yuto;Suzuki, Ken-ichi T.
利用可编程核酸酶进行基因组编辑的最新进展，如锌指核酸酶（ZFNs）、转录激活因子样效应核酸酶（TALENs）和聚集规律间隔短回语重复序列(CRISPR)-Cas系统，促进了热带非洲爪蟾的反向遗传。为了建立使用CRISPR-Cas9分析感兴趣基因的实用工作流程，我们检查了各种实验程序和条件。我们首先通过分析基因型和表型频率以及毒性来比较Cas9蛋白和mRNA注射之间的基因破坏效率。注射带Cas9 mRNA的热带棘猴胚胎，其基因破坏效率与注射Cas9蛋白相当。为了准确评估靶位点的体细胞突变率，使用限制性内切酶或重组Cas9进行扩增子测序和限制性内切片段长度多态性分析。在表现出严重表型的动物中，两种方法估计色素沉着所需的两个靶基因（slc45a2和ltk）的突变率超过90%，这表明靶向体细胞突变在几乎所有创始动物的体细胞中都是双等位引入的。利用异双工迁移率测定，我们还发现脱靶突变的诱导率很低。基于我们的研究结果，我们提出了一种crispr - cas9介导的基因破坏工作流程，用于快速有效地分析热带柽柽树的基因功能。
Recent advances in genome editing using programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system, have facilitated reverse genetics in Xenopus tropicalis. To establish a practical workflow for analyzing genes of interest using CRISPR-Cas9, we examined various experimental procedures and conditions. We first compared the efficiency of gene disruption between Cas9 protein and mRNA injection by analyzing genotype and phenotype frequency, and toxicity. Injection of X.tropicalis embryos with Cas9 mRNA resulted in high gene-disrupting efficiency comparable with that produced by Cas9 protein injection. To exactly evaluate the somatic mutation rates of on-target sites, amplicon sequencing and restriction fragment length polymorphism analysis using a restriction enzyme or recombinant Cas9 were performed. Mutation rates of two target genes (slc45a2 and ltk) required for pigmentation were estimated to be over 90% by both methods in animals exhibiting severe phenotypes, suggesting that targeted somatic mutations were biallelically introduced in almost all somatic cells of founder animals. Using a heteroduplex mobility assay, we also showed that off-target mutations were induced at a low rate. Based on our results, we propose a CRISPR-Cas9-mediated gene disruption workflow for a rapid and efficient analysis of gene function using X.tropicalis founders.