We report model experiments in which simple microinjection of fertilized eggs has been used to effectively perform homology‐directed repair (HDR)‐mediated gene editing in the two Xenopus species used most frequently for research: X. tropicalis and X. laevis. We have used long single‐stranded DNAs having phosphorothioate modifications as donor templates for HDR at targeted genomic sites using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR‐associated protein 9 (CRISPR/Cas9) system. First, X. tropicalis tyr mutant (i.e., albino) embryos were successfully rescued: partially pigmented tadpoles were seen in up to 35% of injected embryos, demonstrating the potential for efficient insertion of targeted point mutations. Second, in order to demonstrate the ability to tag genes with fluorescent proteins (FPs), we targeted the melanocyte‐specific gene slc45a2.L of X. laevis to label it with the Superfolder green FP (sfGFP), seeing mosaic expression of sfGFP in melanophores in up to 20% of injected tadpoles. Tadpoles generated by these two approaches were raised to sexual maturity, and shown to successfully transmit HDR constructs through the germline with precise targeting and seamless recombination. F1 embryos showed rescue of the tyr mutation (X. tropicalis) and tagging in the appropriate pigment cell‐specific manner of slc45a2.L with sfGFP (X. laevis).

中文翻译：

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利用CRISPR / Cas9系统简单地将长单链供体模板进行胚胎注射，即可在热带非洲爪蟾和非洲爪蟾中进行同源性导向的修复。

我们报告中的受精卵注射简单已被用于有效地执行同源定向修复（HDR）介导的基因在这两个编辑模型实验蟾蜍种最常用于研究：十，热带和非洲爪蟾。我们使用簇状规则间隔的短回文重复序列/ CRISPR相关蛋白9（CRISPR / Cas9）系统将具有硫代磷酸酯修饰的长单链DNA用作目标基因组位点HDR的供体模板。首先，热带假单胞菌成功地拯救了突变体（即白化病）胚胎：在多达35％的注射胚胎中发现了部分着色的t，这证明了有效插入靶向点突变的潜力。其次，为了证明能力标记基因与荧光蛋白（FPS），我们有针对性的黑素细胞特异性基因slc45a2.L的非洲爪蟾与标记它超折叠绿色FP（的sfGFP），看到在的sfGFP的马赛克表达高达20％的注入t中的黑色素。通过这两种方法产生的被提高到性成熟，并显示出可以通过精确的靶向和无缝重组成功地通过种系传递HDR构建体。F1胚胎显示出酪氨酸突变（X.热带）和在适当的色素细胞特异性方式标记slc45a2.L用的sfGFP（非洲爪蟾）。