Max Planck Institute for Developmental Biology, Tuebingen 72076, Germany.
Max Planck Institute for Developmental Biology, Tuebingen 72076, Germany
Genetics. 2020 Dec;216(4):947-956. doi: 10.1534/genetics.120.303785. Epub 2020 Oct 15.
A lack of appropriate molecular tools is one obstacle that prevents in-depth mechanistic studies in many organisms. Transgenesis, clustered regularly interspaced short palindromic repeats (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 , and additionally, other species suffer from significantly lower transgenesis efficiencies. 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 drastically different codon usage bias and intron composition. With these findings, we used the species 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 for the detection of CRISPR-edited individuals, which helps considerably to reduce associated time and costs.
缺乏适当的分子工具是阻止许多生物进行深入机制研究的一个障碍。转基因技术、成簇规律间隔短回文重复（CRISPR）相关工程和相关工具是现代生命科学的基础，但它们的应用仍然局限于少数几种模式生物。在线虫门中，除了 之外，只有少数几种其他物种可以进行转基因，此外，其他物种的转基因效率要低得多。我们假设这可能部分是由于受体生物中转基因的不兼容性。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好和内含子组成。有了这些发现，我们使用物种 作为密码子优化和天然内含子添加的概念验证。事实上，我们能够显著提高转基因效率，这一原则可能可用于其他线虫物种。此外，利用改良的转基因，我们在 中开发了一种荧光共注射标记物，用于检测 CRISPR 编辑个体，这极大地减少了相关的时间和成本。
