CRISPR/Cas9-based simple transgenesis in Xenopus laevis
CRISPR/Cas9-based simple transgenesis in Xenopus laevis
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DOI：
10.1016/j.ydbio.2022.06.001
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发表时间：
2022-09-01
影响因子：
2.7
通讯作者：
Mochii, Makoto
中科院分区：
文献类型：
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作者：
Shibata, Yuki;Suzuki, Miyuki;Mochii, Makoto
转基因技术极大地增加了我们的理解，通过活记者成像，以及时空功能的基因的功能，使用损失和获得的功能结构的靶基因的转录调控。在非洲爪蟾物种中，两种成熟的转基因方法，限制性内切酶介导的整合和I-SceI大范围核酸酶介导的转基因，已被用于产生转基因动物。然而，在这两种方法中，供体质粒都被随机整合到非洲爪蟾基因组中。在这里，我们建立了一种新的和简单的基于CRISPR/Cas9的非洲爪蟾靶向转基因技术。在该方法中，将靶向推定的港口位点（转化生长因子β受体2样（tgfbr 2l）基因座）的Cas9核糖核蛋白（RNP）和预设的供体质粒DNA共注射到X的单细胞期胚胎中。光滑。在F0卷曲体中以启动子/增强子特异性方式检测到约10%的忠实报告基因表达。重要的是，在F1后代中观察到有效的种系传递和稳定的转基因表达。该方法的简单性仅需要制备含有tgfbr 2l基因组片段和靶向该位点的Cas9 RNP的供体载体，这是爪蟾实验室中使用的常见实验程序。我们改进的技术允许简单地产生转基因X。laevis，因此有望成为报告基因分析和基因功能分析的有力工具。
Transgenic techniques have greatly increased our understanding of the transcriptional regulation of target genes through live reporter imaging, as well as the spatiotemporal function of a gene using loss- and gain-of-function constructs. In Xenopus species, two well-established transgenic methods, restriction enzyme-mediated integration and I-SceI meganuclease-mediated transgenesis, have been used to generate transgenic animals. However, donor plasmids are randomly integrated into the Xenopus genome in both methods. Here, we established a new and simple targeted transgenesis technique based on CRISPR/Cas9 in Xenopus laevis. In this method, Cas9 ribonucleoprotein (RNP) targeting a putative harbor site (the transforming growth factor beta receptor 2-like (tgfbr2l) locus) and a preset donor plasmid DNA were co-injected into the one-cell stage embryos of X. laevis. Approximately 10% of faithful reporter expression was detected in F0 crispants in a promoter/enhancer-specific manner. Importantly, efficient germline transmission and stable transgene expression were observed in the F1 offspring. The simplicity of this method only required preparation of a donor vector containing the tgfbr2l genome fragment and Cas9 RNP targeting this site, which are common experimental procedures used in Xenopus laboratories. Our improved technique allows the simple generation of transgenic X. laevis, so is expected to become a powerful tool for reporter assay and gene function analysis.