Safe And Controllable Gene Drive System In Drosophila

Advances in the gene editing technology are enabling the engineering of gene drives in insects for pest control.CRISPR/Cas9 makes genetic engineering faster,easier and more efficient.At present,this gene editing technology is widely used to construct gene drive system in many organisms.In this study,we used CRISPR/Cas9 establishe safe and controllable gene drive system both in Drosophila melanogaster and Drosophila suzukii.Meanwhile,we tested the genome editing efficiency of xCas9 at different PAM sites.We also identified the SWD endogenous U6 promoters and compared the gene editing efficiency of each U6 promoter in SWD.These findings may provide tools useful for the development of new genetic strategies for control of agricultural pest.1 Gene drive in Drosophila melanogaster with multiplexed gRNAs targeting female essential geneIn this study,we used the CRISPR/Cas9 gene editing technology establishe a homing-based gene drive system in Drosophila through targeting the tyrosine decarboxylase 2 gene.At the same time,we also constructed three split homing-based gene drive by using transgenic strains that encode three different promoters to express Cas9 in germline of Drosophila.Our results showed that the genomic context of the insertion locus has an effect on the biased inheritance of the gene drive allele.The introduction of exogenous Cas9 improved the gene editing efficiency of CRISPR/Cas9,which is conducive to the biased inheritance of gene drive allele.Using three gRNAs,that could be produced with the tRNA–gRNA architecture simultaneously,targeting the Dm Tdc2 gene increased the range and complexity of resistance selection and reduce the pressure of resistance selection.Octopamine could rescues female mutant Drosophila egg laying with a significant effect of concentration,provides convenience for the factory extensive manufacturing of transgenic gene drive insects.These findings may provide insight into agricultural pest control.2 Homing-based gene drive in Drosophila suzukii with multiplexed gRNAs targeting female essential geneThe invasive spotted wing drosophila,Drosophila suzukii(Matsumura)(Diptera:Drosophilidae)has caused serious economic losses to the fruit industry.In this study,we used the CRISPR/Cas9 gene editing technology establishe a homing-based gene drive system in SWD through targeting the tyrosine decarboxylase 2 gene.In this targeted locus,gene drive allele could achieve super mendelian inheritance.Our results showed that the genetic polymorphism have a significant impact on drive efficacy.We also identified and synthesised the Dsvasa promoter,generating the gene drive SWD transgenic strain expressing Cas9 driven under the Dsvasa promoter.These findings provide a promising start for the application of the homing-based gene drive for control of this important pest.3 Targeted gene disruption by CRISPR/xCas9 system in Drosophila melanogasterIn this study,we applied the CRISPR/xCas9 system to target the white gene in D.melanogaster,testing the genome editing efficiency of xCas9 at different PAM sites.On the GGG PAM site,xCas9 showed less activity than SpCas9.For the non?NGG PAM site TGA,xCas9 could produce DNA cleavage and indel?mediated disruption on the target site,with comparable efficiency to the NGG sites.However,for other non?NGG PAM sites,xCas9 showed no activity.Thus,it is concluded that the genome?editing ability of xCas9 in Drosophila is quite different from that in mammalian cell lines.Therefore,future research is needed to find more robust CRISPR nuclease with increased targeting range for the applications of genome editing in insects.4 Identification and functional analysis of Drosophila suzukii endogenous U6 promotersIn this study,we identified the DsU6 promoter by bioinformatics method in the NCBI genome database.By counting the proportion of individuals with mosaic eyes in G0 flies under the microscope,we can compare the gene editing efficiency of each U6 promoter in SWD.Our results showed that the promoter DsU6-3 had the highest gene editing efficiency.The gene editing efficiency of the promoter DsU6-4 was the lowest.The gene editing efficiency of the Dm U6:3 promoter was lower than that of the DsU6-3 in SWD.These findings expand the range of promoters available to express gRNAs in D.suzukii.