Ohde et al report the first successful development and use of CRISPR/Cas9 in the basally branching insect Thermobia domestica. By co-injecting Cas9 nuclease with sgRNA targeting a T. domestica ortholog of the Drosophila melanogaster white gene (Td-w), the authors created a heritable genetic mutation with a visible marker in this wingless insect.
Insect wings evolved soon after the common ancestor of Ectognatha diverged from other hexapods. T. domestica (common name: firebrat) is a member of Zygentoma, the only wingless order within Ectognatha. Because of its phylogenetic position and ease of rearing, T. domestica could be a powerful model for studying the evolution of insect wings. Firebrat embryos develop over the course of ~12 days, and nymphs take two to four months to reach sexual maturity. Adult females lay throughout the duration of their 3-5 year lifespan.
Before injecting CRISPR reagents into T. domestica embryos, Ohde et al. used Sytox staining to characterize nuclear division and cell migration during the first 24 hours of T. domestica development. Concluding that cellularization probably takes place around 24 hours AEL, the group injected firebrat embyros at 8 hours AEL.
With the goal of inducing indels via NHEJ, the authors injected into two groups two separate sgRNAs targeting aTd-w ortholog pulled out of an unpublished T. domestica transcriptome.
The group reports a hatch rate of approximately one third, of which one third of the emerging nymphs had a loss of pigmentation in the ommatidia, and 80 percent had loss of pigmentation in their body cuticle. Crossing G0 individuals displaying pigmentation defects with wild type individuals yielded offspring with Td-w indels in ~40 percent of genotyped G1 individuals. Among G2 animals, the authors report firebrats homozygous for mutations in Td-w that showed complete lack of eye pigmentation and loss of pigmentation in certain body parts.
The development of CRISPR in the firebrat should be of significant value to researchers working not only on the question of the origin of insect wings but also for those interested in developmental changes that occurred at the dawn of the insect evolution.
Takahiro Ohde, Yusuke Takehana, Takahiro Shiotsuki, Teruyuki Niimi (2018). CRISPR/Cas9-based heritable targeted mutagenesis in Thermobia domestica: A genetic tool in an apterygote development model of wing evolution, Arthropod Structure & Development, 47:362-369,