Perspective on the combined use of an independent transgenic sexing and a multifactorial reproductive sterility system to avoid resistance development against transgenic Sterile Insect Technique approaches

Eckermann, Kolja N.; Dippel, Stefan; KaramiNejadRanjbar, Mohammad; Ahmed, Hassan M. M.; Curril, Ingrid M.; Wimmer, Ernst A.

doi:10.1186/1471-2156-15-s2-s17

Cited by 27 publications

(23 citation statements)

References 72 publications

(89 reference statements)

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“…The latter, however, is the most convenient even though it requires the identification and characterization of suitable promoters. CRISPR/Cas9 holds big promises in the field of insect biotechnology especially for the development of novel pest control strategies, such as reproductive sterility systems based on chromosome shredding (47). To be able to engineer such strategies in D. suzukii, promoters that drive strong expression of gRNAs and other components are of particular importance.…”

Section: Discussionmentioning

confidence: 99%

“…The identification and cloning of the β2t promoter enabled us to generate a spermmarking system in D. suzukii, which provides a tool for basic research in reproductive biology and should help to monitor the success of pest control campaigns in the context of SIT (23)(24)(25)(26). In addition, the β2t promoter can be used in developing novel transgenic pest control approaches (47) for this invasive pest insect.…”

Section: Discussionmentioning

confidence: 99%

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

Ahmed

Hildebrand

Wimmer

2019

Preprint

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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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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

Ahmed

Hildebrand

Wimmer

2019

Preprint

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“…The use of redundant lethality to safe-guard against genetic-breakdown has also been proposed for strains having a Tet-off female-specific lethality system and a male sterility system based upon Q-system repression of sperm-specific endonuclease activity (Eckermann et al, 2014). After activation by quinic acid (by blocking a QS repressor of gene expression), endonuclease activity is expected to shred sperm chromosomes by double-strand breaks, resulting in sterility.…”

Section: An Assessment Of the Potential For Genetic Breakdown Of A Tementioning

confidence: 99%

Enhancing the stability and ecological safety of mass‐reared transgenic strains for field release by redundant conditional lethality systems

Handler

2015

Insect Science

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The genetic manipulation of agriculturally important insects now allows the development of genetic sexing and male sterility systems for more highly efficient biologically-based population control programs, most notably the Sterile Insect Technique (SIT), for both plant and animal insect pests. Tetracycline-suppressible (Tet-off) conditional lethal systems may function together so that transgenic strains will be viable and fertile on a tetracycline-containing diet, but female-lethal and male sterile in tetracycline-free conditions. This would allow their most efficacious use in a unified system for sterile male-only production for SIT. A critical consideration for the field release of such transgenic insect strains, however, is a determination of the frequency and genetic basis of lethality revertant survival. This will provide knowledge essential to evaluating the genetic stability of the lethality system, its environmental safety, and provide the basis for modifications ensuring optimal efficacy. For Tet-off lethal survival determinations, development of large-scale screening protocols should also allow the testing of these modifications, and test the ability of other conditional lethal systems to fully suppress propagation of rare Tet-off survivors. If a dominant temperature sensitive (DTS) pupal lethality system proves efficient for secondary lethality in Drosophila, it may provide the safeguard needed to support the release of sexing/sterility strains, and potentially, the release of unisex lethality strains as a form of genetic male sterility. Should the DTS Prosβ2(1) mutation prove effective for redundant lethality, its high level of structural and functional conservation should allow host-specific cognates to be created for a wide range of insect species.

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“…One possible application for the system in SIT is the development of a reproductive sterility system that targets Cas9 to induce many DSBs at defined loci during spermatogenesis. This could mimic the desired effect of ionizing radiation in generating redundant sterility and at the same time overcome the random action of radiation to all organs, which affects the overall fitness of the sterile males (45).…”

Section: Introductionmentioning

confidence: 99%

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

Ahmed

Hildebrand

Wimmer

2019

Preprint

View full text Add to dashboard Cite

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.

show abstract

Perspective on the combined use of an independent transgenic sexing and a multifactorial reproductive sterility system to avoid resistance development against transgenic Sterile Insect Technique approaches

Cited by 27 publications

References 72 publications

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

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

Enhancing the stability and ecological safety of mass‐reared transgenic strains for field release by redundant conditional lethality systems

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

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