Di Cristina Giulia, Dirksen Elina, Altenhein Benjamin, Büschges Ansgar, Korsching Sigrun I
Institute of Zoology, Faculty of Mathematics and Natural Sciences, University of Cologne, Cologne, Germany.
Institute of Genetics, Faculty of Mathematics and Natural Sciences, University of Cologne, Cologne, Germany.
Sci Rep. 2025 Jan 20;15(1):2584. doi: 10.1038/s41598-025-85911-5.
The parthenogenetic life cycle of the stick insect Medauroidea extradentata offers unique advantages for the generation of genome-edited strains, as an isogenic and stable mutant line can in principle be achieved already in the first generation (G0). However, genetic tools for the manipulation of their genes had not been developed until now. Here, we successfully implement CRISPR/Cas9 as a technique to modify the genome of the stick insect M. extradentata. As proof-of-concept we targeted two genes involved in the ommochrome pathway of eye pigmentation (cinnabar and white, second and first exon, respectively), to generate knockout (KO) mutants. Microinjections were performed within 24 h after oviposition, to focus on the mononuclear (and haploid) stage of development. The KOs generated resulted in distinct eye and cuticle colour phenotypes for cinnabar and white. Homozygous cinnabar mutants showed pale pigmentation of eyes and cuticle. They develop into adults capable of producing viable eggs. Homozygous white KO resulted in a completely unpigmented phenotype in developing embryos that were unable to hatch. In conclusion, we show that CRISPR/Cas9 can be successfully applied to the genome of M. extradentata by creating phenotypically different and viable insects. This powerful gene editing technique can now be employed to create stable genetically modified lines using a parthenogenetic non-model organism.
竹节虫Medauroidea extradentata的孤雌生殖生命周期为基因组编辑品系的产生提供了独特优势，因为原则上在第一代（G0）就可以获得同基因且稳定的突变系。然而，直到现在，用于操纵其基因的遗传工具仍未开发出来。在这里，我们成功地应用CRISPR/Cas9技术来修饰竹节虫M. extradentata的基因组。作为概念验证，我们靶向了参与眼色素沉着的ommochrome途径的两个基因（分别是朱砂基因和白色基因，位于第二和第一外显子），以产生基因敲除（KO）突变体。在产卵后24小时内进行显微注射，以聚焦于单核（单倍体）发育阶段。产生的基因敲除突变体在朱砂基因和白色基因方面表现出明显的眼睛和表皮颜色表型。纯合朱砂突变体的眼睛和表皮色素沉着较浅。它们发育成能够产生有活力卵的成虫。纯合白色基因敲除导致发育中的胚胎出现完全无色素沉着的表型，无法孵化。总之，我们表明，通过创建表型不同且有活力的昆虫，CRISPR/Cas9可以成功应用于M. extradentata的基因组。这种强大的基因编辑技术现在可用于利用孤雌生殖的非模式生物创建稳定的转基因品系。
