Efficient RNA/Cas9-mediated genome editing in Xenopus tropicalis
Efficient RNA/Cas9-mediated genome editing in Xenopus tropicalis
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RNA/Cas9 介导的热带爪蟾基因组高效编辑
DOI：
10.1242/dev.099853
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发表时间：
2014-02-01
期刊：
影响因子：
4.6
通讯作者：
Chen, Yonglong
中科院分区：
文献类型：
--
作者：
Guo, Xiaogang;Zhang, Tiejun;Chen, Yonglong
对于新兴的热带两栖动物遗传模型，靶向基因的破坏依赖于锌指核酸酶(ZFN)或转录激活物样效应核酸酶(TALENS)，这需要复杂的设计和选择或繁琐的构建。因此，简单而高效的基因组编辑工具仍然是该物种非常想要的。在这里，我们报告了RNA引导的Cas9核酸酶导致了我们分析的所有10个热带X.tropicalis基因的精确靶向基因破坏，效率超过45%，很容易达到100%。两个基因座的系统点突变分析表明，间隔区与原间隔区邻近基序(PAM)附近的原间隔区序列之间的完美匹配对于Cas9切割热带疟原虫基因组中的靶点是必不可少的。进一步的研究表明，Cas9系统可以作为非洲爪哇胚胎多重基因组工程的有效工具。对两个基因ptf1a/p48和酪氨酸酶的干扰分析表明，Cas9介导的基因打靶可以促进热带疟原虫胚胎的直接表型评估。最后，以弹性酶-T1、弹性酶-T2或酪氨酸酶为靶点的五只方正蛙显示，目标突变高效地传递到F1胚胎中。综上所述，我们的数据表明Cas9系统是一种简单、高效和可靠的热带管圆线虫多重基因组编辑工具。
For the emerging amphibian genetic model Xenopus tropicalis targeted gene disruption is dependent on zinc-finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), which require either complex design and selection or laborious construction. Thus, easy and efficient genome editing tools are still highly desirable for this species. Here, we report that RNA-guided Cas9 nuclease resulted in precise targeted gene disruption in all ten X. tropicalis genes that we analyzed, with efficiencies above 45% and readily up to 100%. Systematic point mutation analyses in two loci revealed that perfect matches between the spacer and the protospacer sequences proximal to the protospacer adjacent motif (PAM) were essential for Cas9 to cleave the target sites in the X. tropicalis genome. Further study showed that the Cas9 system could serve as an efficient tool for multiplexed genome engineering in Xenopus embryos. Analysis of the disruption of two genes, ptf1a/p48 and tyrosinase, indicated that Cas9-mediated gene targeting can facilitate direct phenotypic assessment in X. tropicalis embryos. Finally, five founder frogs from targeting of either elastase-T1, elastase-T2 or tyrosinase showed highly efficient transmission of targeted mutations into F1 embryos. Together, our data demonstrate that the Cas9 system is an easy, efficient and reliable tool for multiplex genome editing in X. tropicalis.