Improvement and use of CRISPR/Cas9 to engineer a sperm-marking strain for the invasive fruit pest Drosophila suzukii

Abstract Background The invasive fruit pest Drosophila suzukii was reported for the first time in Europe and the USA in 2008 and has spread since then. The adoption of type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) as a tool for genome manipulation provides new ways to develop novel biotechnologically-based pest control approaches. Stage or tissue-specifically expressed genes are of particular importance in the field of insect biotechnology. The enhancer/promoter of the spermatogenesis-specific beta-2-tubulin (β2t) gene was used to drive the expression of fluorescent proteins or effector molecules in testes of agricultural pests and disease vectors for sexing, monitoring, and reproductive biology studies. Here, we demonstrate an improvement to CRISPR/Cas-based genome editing in D. suzukii and establish a sperm-marking system. Results To improve genome editing, we isolated and tested the D. suzukii endogenous promoters of the small nuclear RNA gene U6 to drive the expression of a guide RNA and the Ds heat shock protein 70 promoter to express Cas9. For comparison, we used recombinant Cas9 protein and in vitro transcribed gRNA as a preformed ribonucleoprotein. We demonstrate the homology-dependent repair (HDR)-based genome editing efficiency by applying a previously established transgenic line that expresses DsRed ubiquitously as a target platform. In addition, we isolated the Ds_β2t gene and used its promoter to drive the expression of a red fluorescence protein in the sperm. A transgenic sperm-marking strain was then established by the improved HDR-based genome editing. Conclusion The deployment of the endogenous promoters of the D. suzukii U6 and hsp70 genes to drive the expression of gRNA and Cas9, respectively, enabled the effective application of helper plasmid co-injections instead of preformed ribonucleoproteins used in previous reports for HDR-based genome editing. The sperm-marking system should help to monitor the success of pest control campaigns in the context of the Sterile Insect Technique and provides a tool for basic research in reproductive biology of this invasive pest. Furthermore, the promoter of the β2t gene can be used in developing novel transgenic pest control approaches and the CRISPR/Cas9 system as an additional tool for the modification of previously established transgenes.

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Improvement and Use of CRISPR/Cas9 to Engineer a Sperm-marking Strain for the Invasive Fruit Pest Drosophila suzukii

10.21203/rs.2.12830/v3 ◽

2019 ◽

Author(s):

Hassan M. M. Ahmed ◽

Luisa Hildebrand ◽

Ernst A. Wimmer

Keyword(s):

Genome Editing ◽

Reproductive Biology ◽

Pest Control ◽

Basic Research ◽

Invasive Pest ◽

Agricultural Pests ◽

Drosophila Suzukii ◽

Helper Plasmid ◽

Additional Tool

Abstract Background: The invasive fruit pest Drosophila suzukii was reported for the first time in Europe and the USA in 2008 and has spread since then. The adoption of type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) as a tool for genome manipulation provides new ways to develop novel biotechnologically-based pest control approaches. Stage or tissue-specifically expressed genes are of particular importance in the field of insect biotechnology. The enhancer/promoter of the spermatogenesis-specific beta-2-tubulin (β2t) gene was used to drive the expression of fluorescent proteins or effector molecules in testes of agricultural pests and diseases vectors for sexing, monitoring, and reproductive biology studies. Here, we demonstrate an improvement to CRISPR/Cas-based genome editing in D. suzukii and establish a sperm-marking system. Results: To improve genome editing, we isolated and tested the D. suzukii endogenous promoters of the small nuclear RNA gene U6 to drive the expression of a guide RNA and the Ds heat shock protein 70 promoter to express Cas9. For comparison, we used recombinant Cas9 protein and in vitro transcribed gRNA as a preformed ribonucleoprotein. We demonstrate the homology-dependent repair (HDR)-based genome editing efficiency by applying a previously established transgenic line that expresses DsRed ubiquitously as a target platform. In addition, we isolated the Ds_β2t gene and used its promoter to drive the expression of a red fluorescence protein in the sperm. A transgenic sperm-marking strain was then established by the improved HDR-based genome editing. Conclusion: The deployment of the endogenous promoters of the D. suzukii U6 and hsp70 genes to drive the expression of gRNA and Cas9, respectively, enabled the effective application of helper plasmid co-injections instead of preformed ribonucleoproteins used in previous reports for HDR-based genome editing. The sperm-marking system should help to monitor the success of pest control campaigns in the context of the Sterile Insect Technique and provides a tool for basic research in reproductive biology of this invasive pest. Furthermore, the promoter of the β2t gene can be used in developing novel transgenic pest control approaches. The CRISPR/Cas9 system can be used as an additional tool for the modification of previously established transgenes.

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Improvement and Use of CRISPR/Cas9 to Engineer a Sperm-marking Strain for the Invasive Fruit Pest Drosophila suzukii

10.21203/rs.2.12830/v2 ◽

2019 ◽

Author(s):

Hassan M. M. Ahmed ◽

Luisa Hildebrand ◽

Ernst A. Wimmer

Keyword(s):

Genome Editing ◽

Reproductive Biology ◽

Pest Control ◽

Basic Research ◽

Invasive Pest ◽

Agricultural Pests ◽

Drosophila Suzukii ◽

Helper Plasmid ◽

Additional Tool

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Improvement and Use of CRISPR/Cas9 to Engineer a Sperm-marking Strain for the Invasive Fruit Pest Drosophila suzukii

10.21203/rs.2.12830/v1 ◽

2019 ◽

Author(s):

Hassan M. M. Ahmed ◽

Luisa Hildebrand ◽

Ernst A. Wimmer

Keyword(s):

Genome Editing ◽

Reproductive Biology ◽

Pest Control ◽

Fluorescent Proteins ◽

Basic Research ◽

Invasive Pest ◽

Agricultural Pests ◽

Drosophila Suzukii ◽

Additional Tool

Abstract Background: The invasive fruit pest Drosophila suzukii has been reported for the first time in Europe and USA in 2008 and has spread since then. The adoption of type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) as a tool for genome manipulation has opened the doors for researchers to develop novel biotechnologically based pest control approaches. Stage and or tissue specific genes are of particular importance in the field of insect biotechnology. The enhancer/promoter of the spermatogenesis specific beta-2-tubulin (β2t) gene was used to drive the expression of fluorescent proteins or effector molecules in testes of agricultural pests and diseases vectors for sexing, monitoring and reproductive biology studies. Here, we demonstrate an improvement in the efficiency of CRISPR/Cas-based genome editing in D. suzukii and establish a sperm-marking system.Results: To improve genome editing we isolated and tested the D. suzukii endogenous promoters of the small nuclear RNA gene U6 to drive the expression of a guide RNA and the Ds heat shock protein 70 promoter to express Cas9. As a comparison, we used recombinant Cas9 protein and in vitro transcribed gRNA as a preformed ribonucleoprotein. To evaluate the homology-dependent repair (HDR)-based genome editing efficiency, we used as a target platform a previously established transgenic line that expresses DsRed ubiquitously. In addition, we isolated Ds_β2t gene and used its promoter to drive the expression of a red fluorescence protein in the sperm. A transgenic sperm-marking strain was then established by the improved HDR-based genome editing.Conclusion: The use of the endogenous promoters of D. suzukii U6 and hsp70 genes to drive the expression of gRNA and Cas9, respectively, showed improved efficiency of gene editing compared to previous reports. The sperm-marking system should help to monitor the success of pest control campaigns in the context of the Sterile Insect Technique and provides a tool for basic research in reproductive biology of this invasive pest. Furthermore, the promoter of the β2t gene can be used in developing novel transgenic pest control approaches. The CRISPR/Cas9 system can be used as an additional tool for the modification of previously established transgenes.

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Identification and characterization of four Drosophila suzukii cellularization genes and their promoters

BMC Genetics ◽

10.1186/s12863-020-00939-y ◽

2020 ◽

Vol 21 (S2) ◽

Author(s):

Ying Yan ◽

Syeda A. Jaffri ◽

Jonas Schwirz ◽

Carl Stein ◽

Marc F. Schetelig

Keyword(s):

Control Systems ◽

Pest Control ◽

Embryonic Lethality ◽

Invasive Pest ◽

Economic Damage ◽

Lethal Gene ◽

Gene Promoters ◽

Flanking Sequence ◽

Drosophila Suzukii ◽

Blastoderm Stage

Abstract Background The spotted-wing Drosophila (Drosophila suzukii) is a widespread invasive pest that causes severe economic damage to fruit crops. The early development of D. suzukii is similar to that of other Drosophilids, but the roles of individual genes must be confirmed experimentally. Cellularization genes coordinate the onset of cell division as soon as the invagination of membranes starts around the nuclei in the syncytial blastoderm. The promoters of these genes have been used in genetic pest-control systems to express transgenes that confer embryonic lethality. Such systems could be helpful in sterile insect technique applications to ensure that sterility (bi-sex embryonic lethality) or sexing (female-specific embryonic lethality) can be achieved during mass rearing. The activity of cellularization gene promoters during embryogenesis controls the timing and dose of the lethal gene product. Results Here, we report the isolation of the D. suzukii cellularization genes nullo, serendipity-α, bottleneck and slow-as-molasses from a laboratory strain. Conserved motifs were identified by comparing the encoded proteins with orthologs from other Drosophilids. Expression profiling confirmed that all four are zygotic genes that are strongly expressed at the early blastoderm stage. The 5′ flanking regions from these cellularization genes were isolated, incorporated into piggyBac vectors and compared in vitro for the promoter activities. The Dsnullo promoter showed the highest activity in the cell culture assays using D. melanogaster S2 cells. Conclusions The similarities in the gene coding and 5′ flanking sequence as well as in the expression pattern of the four cellularization genes between D. melanogaster and D. suzukii, suggest that conserved functions may be involved in both species. The high expression level at the early blastoderm stage of the four cellularization genes were confirmed, thus their promoters can be considered in embryonic lethality systems. While the Dsnullo promoter could be a suitable candidate, all reported promoters here are subject to further in vivo analyses before constructing potential pest control systems.

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Immune Response of Drosophila suzukii Larvae to Infection with the Nematobacterial Complex Steinernema carpocapsae–Xenorhabdus nematophila

Insects ◽

10.3390/insects11040210 ◽

2020 ◽

Vol 11 (4) ◽

pp. 210 ◽

Cited By ~ 5

Author(s):

Anna Garriga ◽

Maristella Mastore ◽

Ana Morton ◽

Fernando Garcia del Pino ◽

Maurizio Francesco Brivio

Keyword(s):

Immune Response ◽

Entomopathogenic Nematodes ◽

Humoral Response ◽

Inhibitory Effect ◽

Steinernema Carpocapsae ◽

Invasive Pest ◽

Xenorhabdus Nematophila ◽

Drosophila Suzukii

Entomopathogenic nematodes have been proposed as biological agents for the control of Drosophila suzukii, an invasive pest of small-stone and soft-skinned fruits. Larvae of the fly are susceptible to Steinernema carpocapsae infection but the reaction of immune defenses of the host are unknown. To determine the immune response, larvae were infected with S. carpocapsae and Xenorhabdus nematophila to evaluate the effector mechanisms of both humoral and cellular processes. The symbiont bacteria presented an inhibitory effect on the phenoloxidase cascade with a low level of melanization. Besides, X. nematophila activated the synthesis of putative antimicrobial peptides on the hemolymph of infected larvae. However, those peptides presented a lower antimicrobial activity compared to hemolymph from larvae infected with non-symbiont bacteria. Xenorhabdus nematophila avoided also the phagocytosis response of hemocytes. During in vitro and in vivo assays, S. carpocapsae was not encapsulated by cells, unless the cuticle was damaged with a lipase-treatment. Hemocyte counts confirmed differentiation of lamellocytes in the early phase of infection despite the unrecognition of the nematodes. Both X. nematophila and S. carpocapsae avoided the cellular defenses of D. suzukii larvae and depressed the humoral response. These results confirmed the potential of entomopathogenic nematodes to control D. suzukii.

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A transgenic female killing system for the genetic control of Drosophila suzukii

Scientific Reports ◽

10.1038/s41598-021-91938-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Marc F. Schetelig ◽

Jonas Schwirz ◽

Ying Yan

Keyword(s):

Genetic Control ◽

Control Strategies ◽

Fruit Fly ◽

Female Offspring ◽

Early Gene ◽

Invasive Pest ◽

Drosophila Suzukii ◽

Female Specific

AbstractThe spotted wing Drosophila (Drosophila suzukii) is an invasive pest of soft-skinned fruit crops. It is rapidly transmitted in Europe and North America, causing widespread agricultural losses. Genetic control strategies such as the sterile insect technique (SIT) have been proposed as environment-friendly and species-restricted approaches for this pest. However, females are inefficient agents in SIT programs. Here we report a conditional female-killing (FK) strategy based on the tetracycline-off system. We assembled sixteen genetic constructs for testing in vitro and in vivo. Twenty-four independent transgenic strains of D. suzukii were generated and tested for female-specific lethality. The strongest FK effect in the absence of tetracycline was achieved by the construct containing D. suzukii nullo promoter for early gene expression, D. suzukii pro-apoptotic gene hidAla4 for lethality, and the transformer gene intron from the Mediterranean fruit fly Ceratitis capitata for female-specific splicing. One strain carrying this construct eliminated 100% of the female offspring during embryogenesis and produced only males. However, homozygous females from these FK strains were not viable on a tetracycline-supplemented diet, possibly due to the basal expression of hidAla4. Potential improvements to the gene constructs and the use of such FK strains in an SIT program are discussed.

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The Cellular Innate Immune Response of the Invasive Pest Insect Drosophila suzukii against Pseudomonas entomophila Involves the Release of Extracellular Traps

Cells ◽

10.3390/cells10123320 ◽

2021 ◽

Vol 10 (12) ◽

pp. 3320

Author(s):

Tessa Carrau ◽

Susanne Thümecke ◽

Liliana M. R. Silva ◽

David Perez-Bravo ◽

Ulrich Gärtner ◽

...

Keyword(s):

Immune Responses ◽

Morphological Characteristics ◽

Innate Immune ◽

Invasive Pest ◽

In Vitro Cultivation ◽

Innate Immune Responses ◽

Drosophila Suzukii ◽

Extracellular Traps ◽

First Time

Drosophila suzukii is a neobiotic invasive pest that causes extensive damage to fruit crops worldwide. The biological control of this species has been unsuccessful thus far, in part because of its robust cellular innate immune system, including the activity of professional phagocytes known as hemocytes and plasmatocytes. The in vitro cultivation of primary hemocytes isolated from D. suzukii third-instar larvae is a valuable tool for the investigation of hemocyte-derived effector mechanisms against pathogens such as wasp parasitoid larvae, bacteria, fungi and viruses. Here, we describe the morphological characteristics of D. suzukii hemocytes and evaluate early innate immune responses, including extracellular traps released against the entomopathogen Pseudomonas entomophila and lipopolysaccharides. We show for the first time that D. suzukii plasmatocytes cast extracellular traps to combat P. entomophila, along with other cell-mediated reactions, such as phagocytosis and the formation of filopodia.

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Combining Induced Pluripotent Stem Cells and Genome Editing Technologies for Clinical Applications

Cell Transplantation ◽

10.1177/0963689718754560 ◽

2018 ◽

Vol 27 (3) ◽

pp. 379-392 ◽

Cited By ~ 12

Author(s):

Chia-Yu Chang ◽

Hsiao-Chien Ting ◽

Hong-Lin Su ◽

Jing-Ren Jeng

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Genome Editing ◽

Pluripotent Stem Cells ◽

Basic Research ◽

Congenital Diseases ◽

Novel Technologies ◽

Combined Application ◽

Induced Pluripotent

In this review, we introduce current developments in induced pluripotent stem cells (iPSCs), site-specific nuclease (SSN)-mediated genome editing tools, and the combined application of these two novel technologies in biomedical research and therapeutic trials. The sustainable pluripotent property of iPSCs in vitro not only provides unlimited cell sources for basic research but also benefits precision medicines for human diseases. In addition, rapidly evolving SSN tools efficiently tailor genetic manipulations for exploring gene functions and can be utilized to correct genetic defects of congenital diseases in the near future. Combining iPSC and SSN technologies will create new reliable human disease models with isogenic backgrounds in vitro and provide new solutions for cell replacement and precise therapies.

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Resistance mutation conserved between insects and mites unravels the benzoylurea insecticide mode of action on chitin biosynthesis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1618258113 ◽

2016 ◽

Vol 113 (51) ◽

pp. 14692-14697 ◽

Cited By ~ 70

Author(s):

Vassilis Douris ◽

Denise Steinbach ◽

Rafaela Panteleri ◽

Ioannis Livadaras ◽

John Anthony Pickett ◽

...

Keyword(s):

Genome Editing ◽

Mode Of Action ◽

Management Strategies ◽

Resistance Management ◽

Resistance Mutation ◽

Bioactive Molecules ◽

Agricultural Pests ◽

Homology Directed Repair ◽

A Genome

Despite the major role of chitin biosynthesis inhibitors such as benzoylureas (BPUs) in the control of pests in agricultural and public health for almost four decades, their molecular mode of action (MoA) has in most cases remained elusive. BPUs interfere with chitin biosynthesis and were thought to interact with sulfonylurea receptors that mediate chitin vesicle transport. Here, we uncover a mutation (I1042M) in the chitin synthase 1 (CHS1) gene of BPU-resistantPlutella xylostellaat the same position as the I1017F mutation reported in spider mites that confers etoxazole resistance. Using a genome-editing CRISPR/Cas9 approach coupled with homology-directed repair (HDR) inDrosophila melanogaster, we introduced both substitutions (I1056M/F) in the corresponding flyCHS1gene (kkv). Homozygous lines bearing either of these mutations were highly resistant to etoxazole and all tested BPUs, as well as buprofezin—an important hemipteran chitin biosynthesis inhibitor. This provides compelling evidence that BPUs, etoxazole, and buprofezin share in fact the same molecular MoA and directly interact with CHS. This finding has immediate effects on resistance management strategies of major agricultural pests but also on mosquito vectors of serious human diseases such as Dengue and Zika, as diflubenzuron, the standard BPU, is one of the few effective larvicides in use. The study elaborates on how genome editing can directly, rapidly, and convincingly elucidate the MoA of bioactive molecules, especially when target sites are complex and hard to reconstitute in vitro.

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