Heritable CRISPR/Cas9-mediated targeted integration in Xenopus tropicalis
Heritable CRISPR/Cas9-mediated targeted integration in Xenopus tropicalis
复制标题
热带爪蟾中可遗传的 CRISPR/Cas9 介导的靶向整合。
DOI：
10.1096/fj.15-273425
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发表时间：
2015-12-01
期刊：
影响因子：
4.8
通讯作者：
Chen, Yonglong
中科院分区：
文献类型：
--
作者：
Shi, Zhaoying;Wang, Fengqin;Chen, Yonglong
热带爪蟾是一种新兴的脊椎动物遗传模型。基因敲入方法尚未在该物种中报道。在这里，我们报告说，可遗传的靶向整合可以在这种二倍体青蛙中使用由成簇的规则间隔短回文重复序列（CRISPR）和CRISPR相关蛋白9（CRISPR/Cas9）系统介导的并行切割策略来实现。该策略的关键点是在供体载体中添加Cas9/指导RNA切割位点，允许在体内同时切割染色体靶位点和环状供体DNA。对于测试的3个不同的基因座，都显示出有效的靶向整合，这通过种系传递和Southern印迹分析两者来验证。通过在内含子中设计靶位点，我们能够精确编辑酪氨酸酶编码序列，并从内源性微管蛋白启动子和增强子中获得绿色荧光蛋白的表达。我们无法用T7核酸内切酶I测定法检测脱靶效应。精确编辑X中的蛋白质编码序列。tropicalis扩展了这种二倍体青蛙的用途，例如用于建立研究人类遗传疾病的模型。
Xenopus tropicalis is an emerging vertebrate genetic model. A gene knock-in method has not yet been reported in this species. Here, we report that heritable targeted integration can be achieved in this diploid frog using a concurrent cleavage strategy mediated by the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) system. The key point of the strategy is the addition of a Cas9/guide RNA cleavage site in the donor vector, allowing simultaneous cutting of the chromosomal target site and circular donor DNA in vivo. For the 3 distinct loci tested, all showed efficient targeted integration that was verified by both germ-line transmission and Southern blot analyses. By designing the target sites in introns, we were able to get precise editing of the tyrosinase coding sequence and green fluorescent protein expression from endogenous n-tubulin promoter and enhancers. We were unable to detect off-target effects with the T7 endonuclease I assay. Precise editing of protein coding sequences in X. tropicalis expands the utility of this diploid frog, such as for establishing models to study human inherited diseases.