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<p>The parthenogenetic life cycle of the stick insect <i>Medauroidea extradentata</i> 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 <i>M. extradentata</i>. As proof-of-concept we targeted two genes involved in the ommochrome pathway of eye pigmentation (<i>cinnabar</i> and <i>white</i>, second and first exon, respectively), to generate knockout (KO) mutants. Microinjections were performed within 24&#xa0;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 <i>cinnabar</i> and <i>white</i>. Homozygous <i>cinnabar</i> mutants showed pale pigmentation of eyes and cuticle. They develop into adults capable of producing viable eggs. Homozygous <i>white</i> 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 <i>M. extradentata</i> 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.</p>

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Pioneering genome editing in parthenogenetic stick insects: CRISPR/Cas9-mediated gene knockout in Medauroidea extradentata

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年1月20日

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  • Giulia Di Cristina,
  • Elina Dirksen,
  • Benjamin Altenhein,
  • Ansgar Büschges,
  • Sigrun I. Korsching

摘要

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.