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Genetics Article
Omar Akbari
University of California, San Diego
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
The mosquito Aedes aegypti is the principal vector for arboviruses including dengue/yellow fever, chikungunya, and zika, infecting hundreds of millions of people annually. Unfortunately, traditional control methodologies are insufficient, so innovative control methods are needed. To complement existing measures, here we develop a molecular genetic control system termed precision guided sterile insect technique (pgSIT) in Aedes aegypti. PgSIT uses a simple CRISPR-based approach to generate sterile males that are deployable at any life stage. Supported by mathematical models, we empirically demonstrate that released pgSIT males can compete, suppress, and eliminate mosquitoes in multigenerational population cages. This platform technology could be used in the field, and adapted to many vectors, for controlling wild populations to curtail disease in a safe, confinable, and reversible manner.
Keywords
pgSIT, Aedes aegypti, CRISPR/Cas9, dengue fever, yellow fever, zika, chikungunya, vector control, population elimination
Figure 1
Figure 2
Figure 3
Figure 4
There is NO Competing Interest.
This is a list of supplementary files associated with this preprint. Click to download.
- GUIDEDOA2100081SupplementaryTables.zip
Supplemental Tables Table S1. myo-fem and βTub developmental gene expression data. Ae. albopictus and Ae. aegypti myo-fem and βTub TPM gene expression across different developmental timepoints Table S2. Embryo microinjection and transgenic line generation Table S3. Single gene disruption Table S4. pgSIT cross data Table S5. Life Parameters. Comparisons of life parameters of different mosquito lines. To evaluate the potential fitness costs associated with the pgSIT components, several life-table parameters such as fecundity, larval development time, ♂ insemination capacity, mating competitiveness, and adult survival rate were measured among WT, homozygous gRNAβTub+myo-fem, homozygous Cas9 and heterozygous pgSIT lines (gRNAβTub+myo-fem,+/Cas9,+). Compared to WT, homozygous gRNAβTub+myo-fem, homozygous Cas9, and pgSIT♂’s that produced from maternal Cas9 or paternal Cas9 had no significant differences in larval and pupal survival rate, larval and pupal development time, ♂ insemination ability, and adult survival (p < 0.05). Maternal Cas9 refers to paternal gRNAβTub/+; gRNAmyo-fem/+; maternal Cas9/+, and paternal Cas9 corresponds to maternal gRNAβTub/+; gRNAmyo-fem/+; paternal Cas9/+. Each strain is labeled with a superscript (a-e) and is used to indicate where the significance lies. For example, an ANOVA with a post hoc Tukey’s analysis for ♀ fecundity indicates differences are between wildtype vs gRNA, wildtype vs Cas9, and wildtype vs transheterozygotes. Table S6. pgSIT flight capacity assay. Flight activities were monitored over a 24-hour period using the DAM system. The counts are the number of times the mosquitoes passed the infrared beam. Table S7. Sound attraction assay Table S8. pgSIT♂ sterilize WT ♀’s Table S9. The mean coverage depth from the Nanopore DNA sequencing for all contigs in the genome (2310) and the three plasmids (OA-1067A1: gRNAβTub; OA-1067K: gRNAmyo-fem; and OA-874PA: Nup50-Cas9) as well as normalized coverage based on the global number (Table S12). Transgene coverage ranged from 5.1 to 7.6 and normalized coverage ranged from 0.93 to 1.38. Table S10. Nanopore coverage means Table S11. Mapping Stats for RNA sequencing Table S12. RNAseq expression data Table S13. deseq2_liverpool_males_pgSIT_males.annotations Table S14. deseq2_liverpool_females_pgSIT_females.annotations.xlsx Table S15. deseq2_liverpool_pgSIT.annotations Table S16. Multigenerational population cage data Table S17. Parameters used in Aedes aegypti population suppression model. Table S18. Primer and gRNA sequences
- FileS1ampliconEZseqdata.xlsx
Supplemental Dataset File S1. Amplicon EZ sequencing data.
- SupplementalVideo1.TimelapseofTubmutantandWTtestesandsperm.mp4
Video S1. Timelapse of βTub mutant and WT testes and sperm. βTub mutant and WT testes were imaged at 10X and 63X.
- SupplementalVideo2myofemfemaleseclosingfromcup.mp4
Video S2. myo-fem mutant ♀’s eclosing. Flightless myo-fem mutant ♀’s have abnormal wing postures restricting their escape from rearing cups following eclosion, which reduces survival.
- SupplementalVideo3.Timelapseofmhcmyofemmutantflight.mp4
Video S3. Timelapse of myo-fem mutant flight. Cages consisting of myo-fem mutant ♀’s, myo-fem mutant ♂’s, WT ♀’s, and WT ♂’s were recorded over 5.5 minutes. The cages were occasionally tapped to stimulate movement.
- SupplementalVideo4TimelapseofpgSITandWTmosquitoes.mp4
WT ♂’s were recorded for 5.5 minutes. The cages were occasionally tapped to stimulate movement/flight.
- SupplementalVideo5DAMassayvideo.mp4
Video S5. DAM assay video. Short clip of the DAM assay’s monitor tube in action with a WT ♀ passing the infrared beam during flight.
- SupplementalVideo6.MaleCourtshipAssay.mov
Video S6. Male courtship assay. pgSIT males are strongly attracted to the female flight tone indicating strong mating behavior.
- SupplementlVideo7.mp4
Video S7. Model-predicted impact of releases of pgSIT eggs in Onetahi, Tetiaroa, French Polynesia. Time-series for female Ae. aegypti population density and elimination probability are depicted for four sample release schemes depicted in Figure 4.
- SupplementalFigures.pdf
Supplemental Figures Figure S1. Conservation of target genes in Diptera. Figure S2. Assessment of independent gRNAβTub lines. Figure S3. Assessment of independent gRNAmyo-fem lines. Figure S4. Transmitted light and fluorescent images of mosquito life stages of strains used in this study. Figure S5. Fitness of transheterozygous pgSIT mosquitoes in comparison with WT and parental lines. Figure S6. Illumina NGS-based amplicon sequencing results representing myo-fem and βTub knockout in pgSIT mosquitoes. Figure S7. Determination of transgene copy number using Oxford Nanopore genome sequencing. Figure S8. Integrated genome browser snapshot depicting pgSIT sequencing results for myo-fem. Figure S9. Integrated genome browser snapshot depicting pgSIT sequencing results for βTub. Figure S10. Transcriptional profiling and expression analysis. Figure S11. Scaling pgSIT to control populations of mosquitoes and molecular mechanisms.
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This preprint is under consideration at a Nature Portfolio Journal. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Nature journals have partnered with Research Square to offer In Review, a journal-integrated preprint deposition service.
Genetics Article
Omar Akbari
University of California, San Diego
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 1
Posted 05 Apr, 2021
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
The mosquito Aedes aegypti is the principal vector for arboviruses including dengue/yellow fever, chikungunya, and zika, infecting hundreds of millions of people annually. Unfortunately, traditional control methodologies are insufficient, so innovative control methods are needed. To complement existing measures, here we develop a molecular genetic control system termed precision guided sterile insect technique (pgSIT) in Aedes aegypti. PgSIT uses a simple CRISPR-based approach to generate sterile males that are deployable at any life stage. Supported by mathematical models, we empirically demonstrate that released pgSIT males can compete, suppress, and eliminate mosquitoes in multigenerational population cages. This platform technology could be used in the field, and adapted to many vectors, for controlling wild populations to curtail disease in a safe, confinable, and reversible manner.
Figure 1
Figure 2
Figure 3
Figure 4
There is NO Competing Interest.
This is a list of supplementary files associated with this preprint. Click to download.
- GUIDEDOA2100081SupplementaryTables.zip
Supplemental Tables Table S1. myo-fem and βTub developmental gene expression data. Ae. albopictus and Ae. aegypti myo-fem and βTub TPM gene expression across different developmental timepoints Table S2. Embryo microinjection and transgenic line generation Table S3. Single gene disruption Table S4. pgSIT cross data Table S5. Life Parameters. Comparisons of life parameters of different mosquito lines. To evaluate the potential fitness costs associated with the pgSIT components, several life-table parameters such as fecundity, larval development time, ♂ insemination capacity, mating competitiveness, and adult survival rate were measured among WT, homozygous gRNAβTub+myo-fem, homozygous Cas9 and heterozygous pgSIT lines (gRNAβTub+myo-fem,+/Cas9,+). Compared to WT, homozygous gRNAβTub+myo-fem, homozygous Cas9, and pgSIT♂’s that produced from maternal Cas9 or paternal Cas9 had no significant differences in larval and pupal survival rate, larval and pupal development time, ♂ insemination ability, and adult survival (p < 0.05). Maternal Cas9 refers to paternal gRNAβTub/+; gRNAmyo-fem/+; maternal Cas9/+, and paternal Cas9 corresponds to maternal gRNAβTub/+; gRNAmyo-fem/+; paternal Cas9/+. Each strain is labeled with a superscript (a-e) and is used to indicate where the significance lies. For example, an ANOVA with a post hoc Tukey’s analysis for ♀ fecundity indicates differences are between wildtype vs gRNA, wildtype vs Cas9, and wildtype vs transheterozygotes. Table S6. pgSIT flight capacity assay. Flight activities were monitored over a 24-hour period using the DAM system. The counts are the number of times the mosquitoes passed the infrared beam. Table S7. Sound attraction assay Table S8. pgSIT♂ sterilize WT ♀’s Table S9. The mean coverage depth from the Nanopore DNA sequencing for all contigs in the genome (2310) and the three plasmids (OA-1067A1: gRNAβTub; OA-1067K: gRNAmyo-fem; and OA-874PA: Nup50-Cas9) as well as normalized coverage based on the global number (Table S12). Transgene coverage ranged from 5.1 to 7.6 and normalized coverage ranged from 0.93 to 1.38. Table S10. Nanopore coverage means Table S11. Mapping Stats for RNA sequencing Table S12. RNAseq expression data Table S13. deseq2_liverpool_males_pgSIT_males.annotations Table S14. deseq2_liverpool_females_pgSIT_females.annotations.xlsx Table S15. deseq2_liverpool_pgSIT.annotations Table S16. Multigenerational population cage data Table S17. Parameters used in Aedes aegypti population suppression model. Table S18. Primer and gRNA sequences
- FileS1ampliconEZseqdata.xlsx
Supplemental Dataset File S1. Amplicon EZ sequencing data.
- SupplementalVideo1.TimelapseofTubmutantandWTtestesandsperm.mp4
Video S1. Timelapse of βTub mutant and WT testes and sperm. βTub mutant and WT testes were imaged at 10X and 63X.
- SupplementalVideo2myofemfemaleseclosingfromcup.mp4
Video S2. myo-fem mutant ♀’s eclosing. Flightless myo-fem mutant ♀’s have abnormal wing postures restricting their escape from rearing cups following eclosion, which reduces survival.
- SupplementalVideo3.Timelapseofmhcmyofemmutantflight.mp4
Video S3. Timelapse of myo-fem mutant flight. Cages consisting of myo-fem mutant ♀’s, myo-fem mutant ♂’s, WT ♀’s, and WT ♂’s were recorded over 5.5 minutes. The cages were occasionally tapped to stimulate movement.
- SupplementalVideo4TimelapseofpgSITandWTmosquitoes.mp4
WT ♂’s were recorded for 5.5 minutes. The cages were occasionally tapped to stimulate movement/flight.
- SupplementalVideo5DAMassayvideo.mp4
Video S5. DAM assay video. Short clip of the DAM assay’s monitor tube in action with a WT ♀ passing the infrared beam during flight.
- SupplementalVideo6.MaleCourtshipAssay.mov
Video S6. Male courtship assay. pgSIT males are strongly attracted to the female flight tone indicating strong mating behavior.
- SupplementlVideo7.mp4
Video S7. Model-predicted impact of releases of pgSIT eggs in Onetahi, Tetiaroa, French Polynesia. Time-series for female Ae. aegypti population density and elimination probability are depicted for four sample release schemes depicted in Figure 4.
- SupplementalFigures.pdf
Supplemental Figures Figure S1. Conservation of target genes in Diptera. Figure S2. Assessment of independent gRNAβTub lines. Figure S3. Assessment of independent gRNAmyo-fem lines. Figure S4. Transmitted light and fluorescent images of mosquito life stages of strains used in this study. Figure S5. Fitness of transheterozygous pgSIT mosquitoes in comparison with WT and parental lines. Figure S6. Illumina NGS-based amplicon sequencing results representing myo-fem and βTub knockout in pgSIT mosquitoes. Figure S7. Determination of transgene copy number using Oxford Nanopore genome sequencing. Figure S8. Integrated genome browser snapshot depicting pgSIT sequencing results for myo-fem. Figure S9. Integrated genome browser snapshot depicting pgSIT sequencing results for βTub. Figure S10. Transcriptional profiling and expression analysis. Figure S11. Scaling pgSIT to control populations of mosquitoes and molecular mechanisms.
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