Pioneering Genome Editing in Parthenogenetic Stick Insects: CRISPR/Cas9-Mediated gene knockout in Medauroidea extradentata

Abstract The parthenogenetic life cycle of the stick insect Medauroidea extradentata offers unique advantages for the generation of transgenic lines, as an isogenic and stable transgenic line can in principle be achieved already in the first generation. 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 Medauroidea 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 24h after oviposition, to focus on the monocellular (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, while homozygous white KO resulted in a completely unpigmented phenotype in developing embryos. In conclusion, we show that CRISPR/Cas9 can be successfully applied to the genome of M. extradentata by creating phenotypically different and viable animals. This genetic toolbox can now be employed to create stable genetically modified lines using a parthenogenetic non-model organism.