Three papers recently described successful CRISPR/Cas9 mutagenesis in two species of Spodoptera. Spodoptera litura and S. littoralis.
Bi et al (2016) use Cas9 to create somatic mutations in S. litura in developmental gene abdominal-A, abd-A, and analyzed the resulting larvae and pupae for developmental defects. They describe their egg collection and microinjection strategy. The authors report on their hatch rates and the lethal effects of their specific guide RNA/Cas9 combination. Overall, they got about 20% of their injected embryos to hatch and from those were able to detect mutations frequently. Mutagenesis was so effective that they reported no surviving adults. This was a good example of how efficient mutagensis can be used as a tool for structure function studies without creating transgenic lines.
Zhu et al (2016) take the work a Bi et al (2016) a bit further and mutagenize S. litura‘s pheromone binding protein 3 gene. They report that 87% of the surviving injected embryos and moths were chimeras. The authors targeted the second exon of PBP3 and while they did not confirm the loss of PBP3 protein they did report being unable to detect PBP3 transcripts in homozygous mutants. These homozygotes were tested for their antennal response to three sex pheromone components. Mutant moths responded to all of the components although in some cases the response was quantitatively different from the response of wild type insects.
Finally, Koutroumpa et al (2016) mutagenize the Orco gene of S. littoralis. These authors provide much detail about their injection efforts and genotyping results. Not unlike Zhu et al. (2016) Koutroumpa et al (2016) find that 89.6% of the injected individuals carried Orco mutations and 70% of those transmitted them to the next generation. These authors designed three guide RNAs – two targeting exon 2 and one targeting exon 4. Only one of their guide RNAs was effective at creating mutations – one of the guides targeting exon 2. All of the mutants generated in this study were created with that one guide RNA. Koutroumpa et al (2016) created homozygote mutants and assessed the abilities of their antennae to respond to a variety of plant volatiles as well as sex pheromone. In all cases except proprionic acid the mutant antennae registered no response. These moths had lost their ability to smell.
Although Lepidoptera have been challenging to transform using some transposon technologies, SpCas9 is proving to be very efficient and effective in this group of insects. It will be interesting to see how efficient homology directed repair-based transgene integration will be in these insects.
Koutroumpa, F. A., Monsempes, C., François, M.-C., de Cian, A., Royer, C. et al., 2016 Heritable genome editing with CRISPR/Cas9 induces anosmia in a crop pest moth. Sci Rep 6: 29620doi: 10.1038/srep29620 http://www.nature.com/articles/srep29620 – supplementary-information.
Zhu, G.-H., Xu, J., Cui, Z., Dong, X.-T., Ye, Z.-F. et al., 2016 Functional characterization of SlitPBP3 in Spodoptera litura by CRISPR/Cas9 mediated genome editing. Insect Biochem Mol Biol 75: 1-9doi: http://dx.doi.org/10.1016/j.ibmb.2016.05.006.