Recreating tra-2 Alleles in D. suzukii

Kara Boltz, Ph.D., is a Postdoctoral Research Scholar at North Carolina State University where she is designing and testing conditional CRISPR/Cas9 gene drives for control of spotted wing Drosophila and New World screwworm

Li and Handler (2017) have recently reported using CRISPR/Cas9 to make a temperature-sensitive transformer-2 (tra-2) allele in the pest species Drosophila suzukii by mutating the nucleotides orthologous to the Drosophila melanogaster tra-2ts2 allele. This study demonstrates an important proof of principal that CRISPR/Cas9 can be used to recreate mutant alleles in orthologous genes in organisms lacking extensive genetic tools. Prior to the advent of CRISPR/Cas9 this would have been difficult because a null mutation of the existing gene would have to be made before inserting the mutant transgene.

Image result for tra 2 sex determinationTra-2 is a sex-determination gene involved in regulating female-specific splicing of doublesex pre-mRNA. TRA-2 is required for female differentiation during development as well as for expression of yolk protein (Yp) in adults. In males, TRA-2 is necessary for spermatogenesis. Thus, tra-2 is an attractive target to produce a male-only population of sterile insects for pest control using the sterile insect technique (SIT). Conditional alleles of sex-determination genes could make SIT more efficient by reducing the size of breeding populations, eliminating radiation use, and preventing the co-release of female siblings.

Adult male spotted wing drosophila, Drosophilia suzukii (Matsumura). Photograph by Martin Hauser, California Department of Food and Agriculture.

Li and Handler used CRISPR/Cas9 homology-directed repair to recreate the temperature-sensitive Ds-tra-2ts2 allele in Drosophila suzukii. Flies reared at the permissive temperature (16°C or 20°C) were fertile and developed normally. The D. melanogaster restrictive temperature, 29°C, was lethal for both mutant and wild type D. suzukii. Thus, flies were reared at 26°C or at 16°C followed by a shift to 29°C. XX flies raised at 26°C were intersex with predominately male morphology, which increased when mutants were shifted to higher temperatures as pharate adults. XX pseudo-males did not court or mate females at any temperature, whereas XY mutants mated normally, but were sterile at temperatures over 25.5°C. Finally, Ds-Yp transcripts decreased significantly in mutant XX flies when reared at 29°C. Because D. suzukii could not thrive at higher temperatures, this strategy for SIT may not be optimal for control of these pests. However, the approach could be valuable for control of tropical insect pests.

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Homology Directed Repair. This image is from an excellent mini-review of HDR by Chari Cortez in Addgene’s CRISPR 101 which you will find at http://info.addgene.org/download-addgenes-ebook-crispr-101-2nd-edition

This study has broader implications for medicine and agriculture in situations where recreating an allele in a different organism could facilitate study or use of a mutation. Examples could include understanding the mechanism of a disease-causing allele by putting it into an organism that is easy to grow in the lab, or mutagenizing a model laboratory organism to find beneficial mutations that can then be transferred to the field organism.

Li, J. and A. M. Handler (2017) Temperature-dependent sex-reversal by a transformer-2 gene-edited mutation in the spotted wing drosophila, Drosophila suzukii. Scientific Reports 7, Article number: 12363 doi:10.1038/s41598-017-12405-4

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