Li and Scott report their successful use of Cas9 to knockout two genes in Drosophila suzukii in a recent issue of Biochemical and Biophysical Research Communications.
Drosophila suzukii or the spotted wing Drosophila although a native of Asia has recently become an invasive pest in the US and Europe and this is fueling considerable interest in this insect. What makes D. suzukii a concern is its habit of feeding on otherwise sound fruits such as strawberries and blueberries among others and not rotting or overripe fruit like other species in the genus.
Transferring various genetic technologies developed in and for D. melanogaster to D. suzukii should be fairly straightforward and some basic transgenic technologies have been shown already to be useful in D. suzukii.
Li and Scott chose to target the genes white and Sex Lethal for Cas9 mutagenesis.
Their strategy was to transiently express Cas9 and gene-specific guide RNAs from plasmids injected into preblastoderm embryos. To express Cas9 they used the promoter from the gene vasa from D. melanogaster to regulate expression and to express the gene-specific guide RNAs they used the PolIII promoter from the U6:3 gene of D. melanogaster. These promoters have been previously validated in D. melanogaster and were likely to function in D. suzukii, and they did.
The authors report two successful experiments to create knockouts of white. In the first they produced 44 adults from injected embryos and seem to have recovered at most two events. Only rough estimates of the frequencies of knockouts could be made because of the way the data were reported. In a second slightly larger experiment involving 126 adults from injected embryos seemed to yield about 4 events. The genotypes of white-eyed males was confirmed by DNA sequencing.
For the Sex Lethal knockouts, 132 adults from injected embryos were produced. 27 of those 132 had intersex phenotypes with abnormal genitalia and abnormal ovaries. This was consistent with mutations at Sex Lethal which was confirmed by cloning and sequencing PCR products that overlapped the target site. No germline mutations were reported in this case.
So, Li and Scott have used some genetic components from D. melanogaster and successfully applied them to D. suzukii to implement Cas9-based gene editing in this species. The frequency of mutagenesis seemed low relative to what has been reported in D. melanogaster and the reasons for this was not clear and could be the result of any number of things. I think it is fair to say that there is plenty of room for optimization and Li and Scott’s work will serve as a good starting point.
Given the interest in D. suzukii and the ongoing efforts to devise strategies to control or eliminate it from areas where it is not native there is likely to be more intense study of this insect, and increased demand for functional genomic tools and perhaps the development of genetics-based control or erradication strategies with or without the use of Cas9-based technologies.
Take a look at this manuscript for additional details and insights gained into both of the target genes used in this study.
Li, F., Scott, M. J., 2016 CRISPR/Cas9-mediated mutagenesis of the white and Sex lethal loci in the invasive pest, Drosophila suzukii. Biochem Biophys Res Commun 469: 911-916doi: http://dx.doi.org/10.1016/j.bbrc.2015.12.081.