The lack of appropriate molecular tools is one obstacle that prevents in-depth mechanistic studies in many organisms. Transgenesis, CRISPR-associated engineering, and related tools are fundamental in the modern life sciences, but their applications are still limited to a few model organisms. In the phylum Nematoda, transgenesis can only be performed in a handful of species other than Caenorhabditis elegans, and additionally, other species suffer from significantly lower transgenesis efficiency. We hypothesized that this may in part be due to incompatibilities of transgenes in the recipient organisms. Therefore, we investigated the genomic features of 10 nematode species from three of the major clades representing all different lifestyles. We found that these species show a drastically different codon usage bias and intron composition. With these findings, we used the species Pristionchus pacificus as a proof of concept for codon optimization and native intron addition. Indeed, we were able to significantly improve transgenesis efficiency, a principle that may be usable in other nematode species. In addition, with the improved transgenes, we developed a fluorescent co-injection marker in P. pacificus to detect CRISPR-edited individuals, which helped to considerably reduce associated time and costs.

中文翻译：

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通过密码子优化和原始内含子添加提高Pristionchus线虫的转基因效率和CRISPR相关工具。


缺乏适当的分子工具是阻碍许多生物体深入机理研究的障碍之一。转基因、CRISPR相关工程和相关工具是现代生命科学的基础，但它们的应用仍然仅限于少数模式生物。在线虫门中，转基因只能在除秀丽隐杆线虫以外的少数物种中进行，此外，其他物种的转基因效率明显较低。我们假设这可能部分是由于转基因在受体生物体中的不相容性。因此，我们研究了代表所有不同生活方式的三个主要分支的 10 种线虫物种的基因组特征。我们发现这些物种表现出截然不同的密码子使用偏差和内含子组成。有了这些发现，我们使用Pristionchus pacificus物种作为密码子优化和天然内含子添加的概念证明。事实上，我们能够显着提高转基因效率，这一原理可能适用于其他线虫物种。此外，通过改进的转基因，我们在太平洋对虾中开发了一种荧光共注射标记来检测 CRISPR 编辑的个体，这有助于大大减少相关时间和成本。