Efficient CRISPR-mediated gene targeting and transgene replacement in the beetle Tribolium castaneum
Efficient CRISPR-mediated gene targeting and transgene replacement in the beetle Tribolium castaneum
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DOI：
10.1242/dev.125054
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发表时间：
2015-08-15
期刊：
影响因子：
4.6
通讯作者：
Averof, Michalis
中科院分区：
文献类型：
--
作者：
Gilles, Anna F.;Schinko, Johannes B.;Averof, Michalis
基因编辑技术正在彻底改变我们在许多生物体中进行遗传学的方式。CRISPR/Cas核酸酶已经成为一种高度通用、高效和负担得起的工具，用于靶向基因组中选定的位置。除了在已建立的模式生物中的应用外，CRISPR技术还为广泛物种的遗传干预提供了一个平台，仅限于我们将其运送到细胞和有效选择突变的能力。在这里，我们在一种新兴的昆虫模型和害虫--卡氏拟甲虫身上测试CRISPR技术。我们用简单的方法测试CRISPR/Cas的活性，我们证明了来自Tribolium U6和hsp68启动子的Guide RNAs和Cas9的有效表达，我们还测试了Tribolium中敲除和敲入方法的效率。我们发现55-80%的注射个体携带由非同源末端连接产生的突变(INDELs)，包括马赛克双等位基因敲除；71-100%的人在他们的生殖系中携带此类突变并将其传递给下一代。我们发现CRISPR介导的E-钙粘蛋白基因敲除导致了背部闭合的缺陷，这与RNAi诱导的表型一致。在14%的注射个体中观察到标记基因的同源定向敲入，并通过6%的注射个体传递给下一代。之前在Tribolium上的工作描绘了大量与发育表型和增强子陷阱相关的转基因插入。我们提出了一种有效的方法来重新利用这些插入，通过CRISPR介导的新构建物替换这些转基因。
Gene-editing techniques are revolutionizing the way we conduct genetics in many organisms. The CRISPR/Cas nuclease has emerged as a highly versatile, efficient and affordable tool for targeting chosen sites in the genome. Beyond its applications in established model organisms, CRISPR technology provides a platform for genetic intervention in a wide range of species, limited only by our ability to deliver it to cells and to select mutations efficiently. Here, we test the CRISPR technology in an emerging insect model and pest, the beetle Tribolium castaneum. We use simple assays to test CRISPR/Cas activity, we demonstrate efficient expression of guide RNAs and Cas9 from Tribolium U6 and hsp68 promoters and we test the efficiency of knockout and knock-in approaches in Tribolium. We find that 55-80% of injected individuals carry mutations (indels) generated by non-homologous end joining, including mosaic bi-allelic knockouts; 71-100% carry such mutations in their germ line and transmit them to the next generation. We show that CRISPR-mediated gene knockout of the Tribolium E-cadherin gene causes defects in dorsal closure, which is consistent with RNAi-induced phenotypes. Homology-directed knock-in of marker transgenes was observed in 14% of injected individuals and transmitted to the next generation by 6% of injected individuals. Previous work in Tribolium mapped a large number of transgene insertions associated with developmental phenotypes and enhancer traps. We present an efficient method for re-purposing these insertions, via CRISPR-mediated replacement of these transgenes by new constructs.