ABSTRACT
Originally from Asia, Drosophila suzukii Matsumura is a global pest of economically important soft-skinned fruits. Also commonly known as spotted wing drosophila, it is largely controlled through repeated applications of broad-spectrum insecticides by which resistance has been observed in the field. There is a pressing need for a better understanding of D. suzukii biology and for developing alternative environmentally friendly methods of control. The RNA-guided Cas9 nuclease has revolutionized functional genomics and is an integral component of several recently developed genetic strategies for population control of insects. Here, we describe genetically modified strains that encode three different terminators and four different promoters to express Cas9 robustly in both the soma and/or germline of D. suzukii. The Cas9 strains were rigorously evaluated through genetic crossing to transgenic strains that encode single-guide RNAs targeting the conserved X-linked yellow body and white eye genes. We find that several Cas9/gRNA strains display remarkably high editing capacity. Going forward, these tools will be instrumental for evaluating gene function in D. suzukii and may even provide tools useful for the development of new genetic strategies for control of this invasive species.
Subject(s)
CRISPR-Cas Systems , Drosophila/genetics , Gene Editing/methods , Pest Control, Biological/methods , Animals , CRISPR-Associated Protein 9/genetics , CRISPR-Associated Protein 9/metabolism , Drosophila/pathogenicity , Drosophila Proteins/genetics , Fruit/parasitology , Introduced SpeciesABSTRACT
Engineered reproductive species barriers are useful for impeding gene flow and driving desirable genes into wild populations in a reversible threshold-dependent manner. However, methods to generate synthetic barriers are lacking in advanced eukaryotes. Here, to overcome this challenge, we engineer SPECIES (Synthetic Postzygotic barriers Exploiting CRISPR-based Incompatibilities for Engineering Species), an engineered genetic incompatibility approach, to generate postzygotic reproductive barriers. Using this approach, we create multiple reproductively isolated SPECIES and demonstrate their reproductive isolation and threshold-dependent gene drive capabilities in D. melanogaster. Given the near-universal functionality of CRISPR tools, this approach should be portable to many species, including insect disease vectors in which confinable gene drives could be of great practical utility.
Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , Drosophila melanogaster/genetics , Gene Drive Technology/methods , Genes, Lethal , Genetic Speciation , Population Dynamics , Animals , CRISPR-Associated Protein 9/deficiency , CRISPR-Associated Protein 9/genetics , Drosophila melanogaster/metabolism , Female , Gene Flow , INDEL Mutation , Male , RNA, Guide, Kinetoplastida/genetics , RNA, Guide, Kinetoplastida/metabolism , Reproductive IsolationABSTRACT
CRISPR-based gene-drive systems, which copy themselves via gene conversion mediated by the homology-directed repair (HDR) pathway, have the potential to revolutionize vector control. However, mutant alleles generated by the competing non-homologous end-joining (NHEJ) pathway, resistant to Cas9 cleavage, can interrupt the spread of gene-drive elements. We hypothesized that drives targeting genes essential for viability or reproduction also carrying recoded sequences that restore endogenous gene functionality should benefit from dominantly-acting maternal clearance of NHEJ alleles combined with recessive Mendelian culling processes. Here, we test split gene-drive (sGD) systems in Drosophila melanogaster that are inserted into essential genes required for viability (rab5, rab11, prosalpha2) or fertility (spo11). In single generation crosses, sGDs copy with variable efficiencies and display sex-biased transmission. In multigenerational cage trials, sGDs follow distinct drive trajectories reflecting their differential tendencies to induce target chromosome damage and/or lethal/sterile mosaic Cas9-dependent phenotypes, leading to inherently confinable drive outcomes.
