Progress 09/01/16 to 08/31/21
Outputs
Target Audience:Over the course of the project the PD has given several presentations on the topic of gene drive at scientific conferences and at US and overseas institutions. Stakeholders have been present at some of the conferences. For example, at a conference in 2018 the PD met with representatives of the California cherry board to discuss progress with developing genetic control ofDrosophila suzukii. The PD has also given presentations at the annual BRAG PD meeting. In addition to presentations, the PD leads a postgraduate class at NC State University on "Genetic Pest Management" that is offered in even-numbered years. The class includes a thorough coverage of the many gene drive systems that have been developed over the past several years. More recently, the PD co-teaches a module on "Gene Drive" for a postgraduate "Genetics&Genomics" overview class that is taken by students who want a good background in genetics. Since the last progress report, the PD has not given any presentations in person or by zoom on this project. Changes/Problems:There were two approved no cost extensions for this project. Initially there was a delay in recruiting a postdoctoral fellow. Dr. Bolz began midway through year 1 but only stayed in the lab for a little over one year as she took a position as a senior scientist at a biotech company working on CRISPR/Cas9 editing. It took more than 7 months to find a suitable replacement. Dr. Yadav began midway through year 3. Much of the progress reported was due to the work done by Dr. Yadav. In most of 2020, project spending was delayed as we had limited access to the laboratory. What opportunities for training and professional development has the project provided?The two postdoctoral fellows who worked on this project, Dr. Kara Bolz and Dr. Amarish Yadav had prior experience with Drosophila melanogaster but not D. suzukii. Consequently both required training on how to rear D.suzukii, which is less well adapted to the lab. They were also both trained in how to collect and microinject D. suzukii embryos with plasmid DNA or Cas9 protein-sgRNA complexes. Additionally, they were trained in how to identify transgenic flies using fluorescence microscopy. Lastly, both were trained in how to design sgRNAs and evaluate using an in vitro cleavage assay with Cas9 protein. Dr. Yadav will remain in the laboratory and will present hs work on homing gene drives in D. suzukii at a Gordon conference in 2022. How have the results been disseminated to communities of interest?Progess on the project was presented each year at the BRAG PD meeting. The homing gene drive results in D. suzukii were only obtained in the last year and so we have not had the opportunity to present the data. However, over the course of the project the PD has given several presentations to scientific meetings and to stakeholder groups on genetic control of D. suzukii and C. hominivorax, including the potential of homing gene drive systems. In the final year of the project the following presentations were given: Department of Entomology, Cornell University, virtual presentation via zoom, September 21, 2020. Talk on "Development of genetic systems of insect pests" What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
IMPACT. Spotted wing Drosophila (SWD) (D. suzukii) larvae are significant pests in stone fruits (peach, cherry, and plum) and berries (caneberries, blueberries, and strawberries). D. suzukii is an invasive pest from Asia first discovered in the USA in 2008 and is now widespread. As current insecticide treatments are of limited effectiveness (e.g. weather dependent), non-chemical means for controlling this destructive invasive pest are needed. The New World screwworm (NWS) (Cochliomyia hominivorax) is a devastating pest of warm-blooded animals. Females lay their eggs in open wounds and the hatched larvae eat the animal alive leading to death if untreated. One of the greatest achievement's of the USDA in the 20th century was the development of the sterile insect technique (SIT) for the eradication of C. hominivorax from the U.S.A initially and subsequently from Mexico all of Central America. To prevent re-infestation from South America, sterilized flies are currently being constantly released in a "buffer zone" in eastern Panama and along the border with Colombia. While desirable, it would be very expensive and challenging to use SIT to suppress screwworm in countries where it remains endemic (e.g. South America, Caribbean). In addition, as the screwworm outbreak in the Florida Keys in 2016 demonstrated, the United States remains vulnerable to re-introduction of screwworm. CRISPR/Cas9-mediated gene drives offer a potentially very efficient genetic mechanism for suppressing populations of insect pests. In theory, population suppression could be achieved with at least 100 times fewer released males, which makes a gene drive suppression mechanism economically attractive. To build homing gene drives requires identification of gene promoters active in the male or female germline (i.e. cells in testes or ovaries that will form mature sperm and eggs respectively) to drive production of Cas9. Promoters from genes that make short RNAs such as the U6 genes (U6 RNA is essential for RNA splicing) are needed for expression of guide RNAs (sgRNAs) for Cas9. Lastly, to make a homing gene drive that will be contained in the lab and could not drive in the field we initially worked on making a system that was dependent upon the addition of doxycycline to the diet. However, we found the doxycycline-activated reverse tetracycline transactivator (rtTA) did not provide sufficient robust induction. As an alternative approach we developed a split drive system where the Cas9 gene is present elsewhere in the genome and not part of the homing gene drive gene cassette. This provided effective biological containment while facilitating the evaluation of homing gene drive systems. We report the first homing gene drive system targeting a female essential gene in D. suzukii and discuss the next steps in developing this approach for population suppression. We also report progress on developing a gene drive system in screwworm. We will continue working on homing gene drives in both species. Objective 1. Create conditional female to male transformation and female sterile CRISPR/Cas9 split gene drives in D. suzukii (years 1-2). Completed A split homing gene drive was developed that targeted the female-specific exon of doublesex as targeting the doublesex gene was effective at suppressing a population of mosquitoes in large cages (Kyrou et al [2018] Nature Biotechnol., 36, 1062, PMID:30247490). To do this we first evaluated transgenic lines that express Cas9 in the male and female germlines from the vasa and nanos gene promoters. These are called vasa-Cas9 and nos-Cas9 lines. The level of Cas9 activity in the germline was evaluated by crossing with lines that express sgRNAs that target the white or Sex lethal genes. Cas9-mediated mutagenesis of these genes produces flies with easily scorable phenotypes such as white eyes or XX flies that have both male and female body parts. We next designed and evaluated sgRNAs for the doublesex female exon using an in vitro DNA cleavage assay with Cas9 protein/sgRNA complex. Having identified efficient sgRNAs, a homing gene drive construct was made with a constitutively expressed red fluorescent protein (DsRed) marker gene and a doublesex sgRNA driven by a U6 gene promoter. The DsRed and sgRNA genes were flanked with DNA fragments from the doublesex gene to facilitate homology-directed insertion into the doublesex gene. From embryos that were injected with the DsRed-sgRNA gene construct, four lines were obtained each due to an independent insertion event. Molecular analyses confirmed correct integration at the doublesex gene in all lines. A surprise finding was that in three of the four lines, all females with a single copy of the DsRed-sgRNA gene construct were 100% sterile. RT-PCR analysis confirmed that this was because the XX flies were producing both male and female forms of the doublesex RNA. In the 1C2 line, females with one copy of the DsRed-sgRNA gene construct were fertile and only made the female doublesex RNA. Why insertion of the transgene into the female exon in this line did not cause the RNA splicing machinery to make the male RNA splice is unclear. Consequently, only in the 1C2 line can homing gene drive can occur in both sexes. To measure gene drive, nos-Cas9 or vasa-Ca9 virgin females were crossed with males that carried a single copy of the DsRed-sgRNA construct inserted into the doublesex female exon. Male offspring were collected that carried the nos-Cas9 or vasa-Ca9 gene and the DsRed-sgRNA gene. The males were crossed with wild type virgin females. The offspring of the crosses were scored for the number that had red fluorescence (from the DsRed transgene) and the number that had no fluorescence. If there was no drive, 50% of the offspring would show red fluorescence. When crossed with nos-Cas9, the percentage of DsRed positive offspring ranged from 59% (3A3 dsx-sgRNA line) to 70% (1C2 dsx-sgRNA line), thus showing successful homing gene drive. For population suppression it would be desirable if the frequency of drive was higher. To improve drive we are testing new Cas9 gene constructs that should be more active in the germline and a new DsRed-sgRNA gene construct that expresses multiple sgRNA targeting the female exon of doublesex. Objective 2. Create conditional female to male transformation CRISPR/Cas9 split gene drives in C. hominivorax (years 1-2). 50% complete We assembled and annotated the genome for C. hominivorax (Scott et al [2020] Communications Biology, 3,424, https://doi.org/10.1038/s42003-020-01152-40). Genes that were active in the germline that could be a source of promoters for driving Cas9 were identified. These included the C. hominivorax zero population growth, vasa, nanos, beta2-tubulin and bag of marbles genes. Gene constructs were made with each of these promoters driving fluorescent protein marker genes or the tetracycline transactivator (tTA). The latter provides a sensitive method for detecting promoter activity in the germline as we previously made a tTA-activated DsRed reporter strain. Transgenic lines were made with a vasa promoter-GFP gene construct. Unfortunately, the vasa promoter appears to be inactive as none of the transgenic lines showed any GFP expression. Assessment of the other germline promoters is underway. Five U6 genes were identified in the genome. Gene constructs have been made with each promoter driving expression of a sgRNA that targets Cas9 to the DsRed gene. The U6 gene promoters will be compared by injecting embryos with Cas9 protein and plasmid DNA carrying a U6-sgRNA construct. The most active U6 promoter would produce the highest level of Cas9 cleavage of the DsRed gene in embryos. Once the germline and U6 promoters have been evaluated, we will make a split homing gene drive system targeting the doublesex female exon. Objectives 3 and 4. Not completed We were unable to begin these objective due to delays in completing objectives one and two.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Long, K.C., Alphey, L., Annas, G.J., Bloss, C.S., Campbell, K.J., Champer, J., Chen, C.H., Choudhary, A., Church, G.M., Collins, J.P., Cooper, K.L., Delborne, J. A., Edwards, O.R., Emerson, C.I., Esvelt, K., Evans, S.W., Friedman, R.M., Gantz, V.M., Gould, F., Hartley, S., Heitman, E., Hemingway, J., Kanuka, H., Kuzma, J., Lavery, J.V., Lee, Y., Lorenzen, M., Lunshof, J.E., Marshall, J.M., Messer, P.W., Montell, C., Oye, K.A., Palmer, M.J., Papathanos, P.A., Paradkar, P.N., Piaggio, A. J., Rasgon, J.L., Raai?, G., Rudenko, L., Saah, J.R., Scott, M.J., Sutton, J.T., Vorsino, A.E. & Akbari, O.S. (2020). Core commitments for field trials of gene drive organisms. Science 370: 1417-1419. DOI: 10.1126/science.abd1908
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Progress 09/01/19 to 08/31/20
Outputs
Target Audience:The PD gave several presentations given during the reporting period that included our work on developing gene drive systems in SWD and screwworm. The audience for these presentations were practicing scientist that work in the field of genetics. The invited talks in Europe were given while the PD was on a one semester sabbatical at the University of Zurich. No travel cost were charged to this project. The talks were: Annual meeting of the American Veterinary Medical Association (AVMA), virtual convention, August 21 2020. Invited talk on "Genetic control of the New World screwworm". Developmental Biology at the Johann Friedrich Blumenbach Institute of Zoology and Anthropology, Georg August University, Göttingen, Germany, (May 28, 2020) "Development of genetic/genomic resources for basicinvestigations and for genetic control of blow flies". talk was given via zoom. Max Planck Institute of Immunology and Epigenetics, Freiburg im Breisgau, Germany, October 18, 2019. Invited talk on "Development of genetic systems for control of insect pests". · Groningen Institute for Evolutionary Life Sciences (GELIFES) in Groningen, Netherlands, October 3, 2019. Invited talk on "Development of genetic systems for control of insect pests". · Joint meeting of the Associazione Genetica Italiana (AGI) and Società Italiana di Mutagenesi Ambientale e Genomica (SIMAG), Cortona, Italy, 26-28 September 2019. Invited talk on "Development of genetic systems for control of insect pests". Institute of Molecular Life Sciences, University of Zürich, Switzerland, September 17, 2019. Invited talk on "Development of genetic systems for control of insect pests". Changes/Problems:COVID_19 has significantly impacted our progress on this project. The lab was shutdown in mid-March 2020. We were only able to maintain existing strains until limited was access was granted in the summer. At the end of the reporting period, personnel were working each day but on time-limited and personnel-limited shifts ( no more than 1 person pre 200 sq ft). In addition, with the onset of covid-19 the USDA research laboratory in Panama was only able to maintain existing strains of C. hominivorax with very limited capacity to undertake new research. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Objective 3. Evaluate the potential for population suppression in cage experiments under permissive and non-permissive conditions (years 2-3). Work on this objective can now begin in the next reporting period using the doublesex mutant made in the previous reporting period. Rather than a conditional drive regulated by tetracycline we will test for drive using a "split" system with the Cas9 gene not part of the homing gene construct. We changed to working on split gene drives as we previously reported that the rtTA tetracycline-On system did not appear to be a good approach for controlling Cas9 activity in flies.
Impacts
What was accomplished under these goals?
IMPACT. Spotted wing Drosophila (SWD) (D. suzukii) larvae are significant pests in stone fruits (peach, cherry, and plum) and berries (caneberries, blueberries, and strawberries). D. suzukii is an invasive pest from Asia first discovered in the USA in 2008 and is now widespread. As current insecticide treatments are of limited effectiveness (e.g. weather dependent), non-chemical means for controlling this destructive invasive pest are needed. The New World screwworm (NWS) (Cochliomyia hominivorax) is a devastating pest of warm-blooded animals. Females lay their eggs in open wounds and the hatched larvae eat the animal alive leading to death if untreated. One of the greatest achievement's of the USDA in the 20th century was the development of the sterile insect technique (SIT) for the eradication of C. hominivorax from the U.S.A initially and subsequently from Mexico all of Central America. To prevent re-infestation from South America, sterilized flies are currently being constantly released in a "buffer zone" in eastern Panama and along the border with Colombia. While desirable, it would be very expensive and challenging to use SIT to suppress screwworm in countries where it remains endemic (e.g. Brazil, Colombia, Cuba, Jamaica). Cas9-mediated gene drives offer a potentially very efficient genetic mechanism for suppressing populations of insect pests. In theory, a gene drive could effectively suppress insect populations with a single release of 1 male for every 10 fertile males in the region. That is, 100 times fewer males than SIT, which makes a gene drive suppression mechanism economically attractive. However, they pose a challenge for regulators as, by their very nature, they are designed to not be contained. The overall goal of our study is to engineer novel conditional (i.e. with an ON/OFF switch) contained gene drives and evaluate efficacy and safeguards in D. suzukii and C. hominivorax. The drives will target the genes essential for female development or reproduction. This research will provide information for regulators on the use of conditional gene drives to suppress an insect population. With additional engineering, it would be possible to develop strains containing drives that could be suppressed in the laboratory but would drive in the field. Such strains could provide a very efficient mechanism for suppression of these major insect pests. To build conditional drives requires identification of gene promoters active in the male or female germline (i.e. cells in testes or ovaries that will form mature sperm and eggs respectively). Promoters from genes that make short RNAs such as the U6 genes (U6 RNA is essential for RNA splicing) are needed for expression of guide RNAs (gRNAs) for Cas9. Lastly, to make the system conditional on the addition of doxycycline, the most active form of the reverse tetracycline transactivator (rtTA) in flies needed to be identified. rtTA, a transcription activator, would be used to control expression of Cas9 in the germline. Objective 1. Create conditional female to male transformation and female sterile CRISPR/Cas9 split gene drives in D. suzukii (years 1-2). Estimate 75% complete The doublesex gene is essential for female development. Indeed, it has been reported that a homing gene drive targeting the doublesex gene was effective at suppressing a population of mosquitoes in large cages (Kyrou et al [2018] Nature Biotechnol., 36, 1062, PMID:30247490). Previously, we reported the identification of the D. suzukii doublesex gene and the evaluation of gRNAs for the female-specific exon. We then made a homing gene construct consisting of the optimal gRNA driven by a U6 promoter, a constitutively expressed fluorescent protein marker gene and left and right homology arms. In this reporting period, the construct was injected into D. suzukii embryos and transgenics identified by expression of the marker gene. Molecular analysis has confirmed precise insertion of the gene construct into the doublesex gene. We had predicted that homozygous females would be sterile. Somewhat surprisingly, our preliminary data indicate a high level of female sterility in heterozygous females. Transgenic males were fully fertile and viable. If subsequent analysis confirms the sterility of heterozygous females, then homing drive will be limited to males. We previously reported the evaluation of transgenic strains that express Cas9 in the male and female germline from the nanos and vasa gene promoters. For split gene drive experiments, an X-linked nanos-cas9 line (line #36X) that shows high Cas9 activity in the germline but little in the soma will be used. In these experiments, nos-Cas9 virgin females will be crossed to heterozygous doublesex mutant males that carry the DsRed marker-gRNA gene cassette in the female exon. Male offspring will be collected and crossed with wild type virgin females. If there is gene drive, significantly more than 50% of the offspring will inherit that mutant doublesex gene with the DsRed marker-gRNA gene cassette We are collaborating with Dr. Omar Akbari's lab at the University of San Diego on evaluation of transgenic D. suzukii strains that express Cas9 and gRNAs. During this reporting period, the Akbari lab shipped a transgenic strain that expresses a gRNA for the D. suzukii yellow gene. D. suzukii homozygous for mutations in yellow develop a yellow body color. Thus, the gRNA strain provides an additional independent confirmation on the activity of the Cas9 driver lines. At the end of the reporting period we had begun crossing the yellow gRNA strain with our Cas9 lines. We anticipate that the experiments will be completed by the end of 2020. The plan is to publish a paper with the Akbari lab on Cas9 and gRNA strains for efficient gene editing in D. suzukii. Objective 2. Create conditional female to male transformation CRISPR/Cas9 split gene drives in C. hominivorax (years 1-2). 40% complete Progress on this objective has been significantly disrupted due to covid-19. The research laboratory in Panama was essentially shutdown in early March with essential personnel only able to maintain existing strains. With limited lab access due to covid-19 we have chosen to focus our time mostly on objective 1. For objective 2, the identification of a germline promoter that can drive Cas9 expression remains an essential step. We previously reported that the C. hominivorax vasa promoter-GFP-vasa 3'UTR gene construct was inactive in transgenic blow flies. As part of the C. hominivorax genome project we manually annotated other candidate germline promoters (Scott et al, 2020). As the nanos promoter has proven to be germline specific in Drosophila and mosquitoes we began construction of the C. hominivorax nanos promoter driving tTA casette. Expression of tTA in the germline will be assessed by crossing with a tetO-DsRed reporter strain made previously. Objective 3. Evaluate the potential for population suppression in cage experiments under permissive and non-permissive conditions (years 2-3). 0% complete. No progress on this objective during the reporting period. Objective 4. Evaluate the potential for stopping a drive by releasing flies that are immune to Cas9 (years 2-3). 0% complete. Work on this objective cannot begin until objective 2 is completed. Given progress on objective 2, this is unlikely to begin in the last reporting period.
Publications
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Progress 09/01/18 to 08/31/19
Outputs
Target Audience:The PD gave a presentation at the "Sustainable SWD Management for US Fruit crops" meeting in Raleigh, North Carolina, February 21-22, 2019. The title of the talk was "Development and evaluation of GM strains for genetic control of spotted wing Drosophila (SWD)". The meeting was attended by Drosophila suzukii researchers and stakeholders. The PD also gave updates on our work every quarter to stakeholders who have an interest in new tools for SWD management. The conference calls are organized by my colleague at NC State, Dr. Hannah Burrack. The PD gave a poster at the Project Director's meeting for the Biotechnology Risk Assessment Grants (BRAG) Program (June 6, 2019) in Riverdale, MD. The title of the poster was "Development and evaluation of safeguards for conditional suppressive gene drives for spotted wing Drosophila and the New World screwworm ". The PD had discussions with regulators and other participants at the meeting regarding the current state of development gene drive systems in agricultural pests. Other conferences presentations given during the reporting period that included our work on developing gene drive systems in SWD and screwworm: Annual conference of the Australian Society for Parasitology, Adelaide, Australia, 8-11 July, 2019. Invited plenary talk on "Development of genetic systems for control of New World screwworm and sheep blowfly". Gene Drive Modeling Conference, Washington DC, June 11-12 2019. Invited talk on "Potential Applications of Gene Drives for Control of Agricultural Insect Pests". Advances in biotechnology and their potential use in the eradication of the New World screwworm, Montevideo, Uruguay, 15-16 January, 2019. Two invited talks "Genetic control of screwworm using transgenic male-only strains" and "Screwworm Cas9 gene drive". Annual meeting of the Entomological Society of America, Vancouver, Canada, November 11-14, 2018. One talk: "Development of Gene Drive Systems for Control of Spotted Wing Drosophila and New World Screwworm" "3rd Meeting of the Gene Drive Research Sponsors and Supporters Forum", Montreal, Canada, September 11-13, 2018. Presentation "Development of gene drive systems for control of spotted wing Drosophila and New World screwworm". The PD co-taught a class on "Genetic Pest Management" in the fall semester of 2018. The course covers all genetic approaches for control of insect pests starting with the classical sterile insect technique and ending with efficient but self-sustaining gene drive systems. The course was taken by 7 doctoral students from several departments in the College of Agriculture. Changes/Problems:The senior postdoctoral fellow, Kara Bolz resigned in May 2018 to take the position of chief scientist in a small startup company in Florida. This was unfortunate as, after a slow start, she was beginning to make progress on the project (e.g. developed rapid assay for gRNA testing, CRISPR with long single stranded oligonucleotides). The position was listed and short-listed applicants interviewed. We hired Dr. Amarish Yadav who at that time was completing his doctoral studies on apoptosis in Drosophila melanogaster. Dr Yadav arrived at the end of the 2018. He has been learning how to work with D. suzukii, how to make gene constructs and how to make transgenic Drosophila. I expect his productivity will improve in the second year. As noted in the previous report, there was not sufficient remaining funds to employ a postdoctoral fellow for 2 years, which is what we have found is the minimum period needed to recruit qualified applicants. To be able to offer a two-year position, a fraction of his salary (<50%) comes from other sources. Thus Dr. Yadav will be spending most, but not all, of his time on the BRAG project. What opportunities for training and professional development has the project provided?The new hire for this project, Dr Amarish Yadav, has been trained in embryo microinjection, DNA cloning and how to raise and cross Drosophila suzukii. Dr. Yadav's PhD was on Drosophila melanogaster. He has considerable experience with rearing and crossing D. melanogaster lines and examining protein distribution in tissues using confocal microscopy. How have the results been disseminated to communities of interest?As discussed above, the PD presentations at several conferences. All of the talks included work on gene drives. The PD also presented progress on D. suzukii projects at quarterly national stakeholder meetings. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Create conditional female to male transformation and female sterile CRISPR/Cas9 split gene drives in D. suzukii (years 1-2). Make transgenic lines of D. suzukii with homing gene constructs targeting doublesex and other genes that disrupt female fertility (e.g. yellow-g) Cross homing line with lines that express Cas9 in the germline. Monitor homing frequency by conversion of heterozygote to homozygote in the germline. Objective 2. Create conditional female to male transformation CRISPR/Cas9 split gene drives in C. hominivorax (years 1-2). Identify the best gRNA for selected target genes using the in vitro DNA cleave assay. Make transgenic lines with the bicoid promoter gene construct. Make additional germline promoter constructs (e.g. orthologs of nanos, bag of marbles)
Impacts
What was accomplished under these goals?
Objective 1. Create conditional female to male transformation and female sterile CRISPR/Cas9 split gene drives in D. suzukii (years 1-2). Estimate 50% complete Before assembling the final gene drives we must test the efficacy of guide RNAs (gRNAs) as it is well known that some gRNAs are more efficient than others at directing the Cas9 nuclease to the targeted gene. We previously reported development of a rapid in vitro (i.e. test tube) DNA cleavage assay with purified DNA template, Cas9 protein and gRNA. It has been recently reported that a homing gene drive targeting the doublesex gene was effective at suppressing a population of mosquitoes in large cages (Kyrou et al [2018] Nature Biotechnol., 36, 1062, PMID:30247490). Consequently, we identified the D. suzukii doublesex gene and then designed and tested gRNAs for the female-specific exon. We then made a homing gene construct consisting of the optimal gRNA driven by a U6 promoter, a constitutively expressed fluorescent protein marker gene and left and right homology arms. The construct will be injected into D. suzukii embryos and transgenics identified by expression of the marker gene. If successful, the construct will target and inactivate the doublesex gene, which will make the females sterile. The construct will not drive as it lacks a Cas9 gene. Rather, drive potential can be assessed by crossing to our existing strains that express Cas9 in the germline. We previously reported the evaluation of transgenic strains that express Cas9 in the male and female germline from the nanos and vasa gene promoters. We are collaborating with Dr. Omar Akbari's lab at the University of San Diego on evaluation of transgenic D. suzukii strains that express Cas9 and gRNAs. As part of this collaboration, they shipped their strains that express Cas9 from the BicC and dhd promoters. We evaluated their strains by crossing to our transgenic strains that express a gRNA for the Sex lethal (Sxl) gene. Sxl is the master gene that determines female sex and X chromosome dosage compensation in Drosophila. In D. melanogaster, Sxl directly regulates ovary development through repression of the bag of marbles gene. We crossed a Sxl gRNA line with lines that express Cas9 in the germline from three promoters (nanos, vasa, BicC) to determine which is the best for producing Cas9 for homing gene drive. The results are summarized below: -nos-Cas9. The sex ratio was normal in offspring of the cross and females had normal external morphology. However, females were sterile. Upon dissection we found that the females had not developed ovaries. This experiment shows that nos-cas9 is confined to the germline and that Cas9-mediated knockout of the both Sxl alleles in the germline is very efficient. -vasa-Cas9. Offspring showed a slight male bias (60% males). All female offspring has deformed ovipositors and were sterile. Since the ovipositor is a somatic tissue, this experiment confirms that vasa-Cas9 had significant activity in somatic cells and thus would not be ideal for gene drive. -BicC-Cas9. Offspring showed a slight male bias (60% males). About 20% of female offspring has deformed ovipositors indicating that BicC-Cas9 had some activity in somatic cells and so would not be ideal for gene drive. We previously reported that we are evaluating two reverse tetracycline trans-activators (rtTAs) G3 and M2, that only bind to DNA in the presence of tetracycline or doxycycline. None of the rtTAs appeared to activate ZsGreen reporter gene in testes. Consequently, we make rtTA autoregulated overexpression constructs and evaluated in transgenic Drosophila melanogaster. The prediction was that, if the rtTAs are functional, rtTA protein levels would accumulate to very high levels when the flies are raised on diet with tetracycline/doxycycline. High levels of tTA are lethal for a wide range of insect species. On standard diet the % pupal hatch was comparable to the parental wild type strain. However, in the presence of 100 ug/mL doxycycline, 85% of the pupae failed to hatch for both G3 and M2 overexpression gene construct. Thus both versions of rtTA appear to be effective and of similar activity in Drosophila. We previously reported using Cas9, a gRNA and a long single stranded oligonucleotide template to insert an attP recombination site into a specific location in the D. suzukii genome. This was one of the last experiments done by Dr. Kara Bolz, the former postdoctoral fellow who worked on this project. The strain was not homozygous when she resigned. It was kept in culture for 8 months until the current postdoctoral fellow arrived. He found that about 1 in 20 flies still carried the attP insertion. At the end of the reporting period the strain was not yet 100% homozygous for the attP site. After we make the strain homozygous, we will test if the attP site can be used for site specific recombination and thus allow us to test various gene constructs at the same location in the genome. This is important as the site of transgene integration can have a large impact on the level of gene expression. Objective 2. Create conditional female to male transformation CRISPR/Cas9 split gene drives in C. hominivorax (years 1-2). 40% complete In the reporting period we continued our effort to isolate and characterize screwworm U6 and germline promoters to make homing gene drive constructs. We previously reported that transgenic Lucilia lines have been made with a vasa promoter-GFP-vasa 3'UTR gene construct. If active, vasa promoter should drive GFP expression in ovaries. Also, eggs deposited by the female would glow green under blue light due to GFP expression. Unfortunately, none of the transgenic lines showed any GFP expression. We conclude that the vasa promoter is not active. We then made new gene constructs with the putative promoter from the bicoid gene driving GFP expression. Next the construct will be injected into Lucilia embryos and transgenic lines screened for green fluorescence. To assay for rtTA function, a transgenic line was made by inserting a CG14427 promoter-rtTA M2 construct into a specific site in the genome using bacterial phiC31 recombinase. The CG14427 promoter is active in the early embryo. We performed a molecular analysis to determine if the construct had inserted at the specific site in the genome. This was the location of an attP sequence that is recognized by the phiC31 recombinase. Surprisingly, the transgene had inserted at another site in the genome. It is possible that the phiC31 recombinase is recognizing a DNA sequence similar to the attP site. A tetO-hid gene that is activated by rtTA was located adjacent to the attP site. If rtTA was expressed in the embryo, activation of the hid gene would cause female embryos to die. However, we observed no lethality in the presence or absence of doxycycline (activator of rtTA). It appears that the rtTA protein has low activity in blowflies and thus would not appear be a suitable choice for regulating Cas9 expression for a conditional drive system. As mentioned above, we have made a homing gene drive construct targeting the doublesex gene in D. suzukii. This construct will be evaluated in the next reporting period. Objective 3. Evaluate the potential for population suppression in cage experiments under permissive and non-permissive conditions (years 2-3). 0% complete. Work on this objective cannot begin until objective 1 or 2 is completed. Objective 4. Evaluate the potential for stopping a drive by releasing flies that are immune to Cas9 (years 2-3). 0% complete. Work on this objective cannot begin until objective 1 or 2 is completed.
Publications
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Progress 09/01/17 to 08/31/18
Outputs
Target Audience:The PD gave a presentation at the "Sustainable SWD Management" meeting in Portland, Oregon, February 5-6, 2018. The title of the talk was "Progress towards genetic control of spotted wing Drosophila". The meeting was attended by D. suzukii researchers and stakeholders. The PD had several discussions with stakeholders (eg California cherry board) who asked for more collaboration between groups working on SWD genetics. As a consequence, we shipped D. suzukii transgenic strains that express guide RNAs to Omar Akbari's lab at the University of San Diego. They have used the strains to evaluate their transgenic lines that express Cas9. From this effort we are planning a joint publication. An APHIS permit was obtained for all shipments. The PD gave a poster at the Project Director's meeting for the Biotechnology Risk Assessment Grants (BRAG) Program (May 22, 2018) in Riverdale, MD. The title of the poster was "Development and evaluation of safeguards for conditional suppressive gene drives for spotted wing Drosophila and the New World screwworm ". The PD had several discussions with regulators at the meeting regarding our SOP for working with transgenic D. suzukii. Changes/Problems:The postdoctoral fellow resigned in May to take a position at a small startup biotech company in Florida. They will be the main person responsible for developing a Cas9-based approach for control of citrus greening. In July we advertised for a replacement for the postdoctoral fellow. There is not sufficient remaining funds to employ a postdoctoral fellow for 2 years, which is what we have found is the minimum period needed to recruit qualified applicants. To be able to offer a two-year position, 25% of the salary will come from a NIFA-SCRI grant that is focused on developing male-only SWD strains for genetic control programs. Thus the replacement fellow will be spending most, but not all, of their time on the BRAG project. Prior to resigning, the fellow's progress was slowed by unexpected technical difficulties in developing an assay for gRNA efficiency. The DNA melting assay used for other organisms proved to be unreliable for SWD. We think this is because of the large level of natural DNA polymorphism present in our SWD lab stock, which was recently (<12 months) established with flies caught in North Carolina. The in vitro DNA cleavage assay that was developed produced quick results and led to the identification of optimal gRNA for most targets. This information will be used in developing U6 promoter gRNA constructs. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? Objective 1. Create conditional female to male transformation and female sterile CRISPR/Cas9 split gene drives in D. suzukii (years 1-2). Transgenic lines will be made with U6-gRNA constructs. These will be with the best gRNAs identified using the in vitro DNA cleavage assay. The lines will be made either with piggyBac or phiC31 recombinase. For the latter we will use the recently generated attP line. Transgenic rtTA overexpression lines will be examined for rtTA expression in the presence or absence of doxycycline. This analysis will show which is the most active rtTA in Drosophila. Assemble conditional gene drive constructs containing the best rtTA driven by the nanos promoter, a tetO-Cas9 cassette (made previously), all flanked by "homology arms" that match the DNA sequence to either side of the Cas9 cut site. Make transgenic lines with the conditional Cas9 expression construct by injecting embryos that express the appropriate gRNA. Objective 2. Create conditional female to male transformation CRISPR/Cas9 split gene drives in C. hominivorax (years 1-2). Identify the best gRNA for selected target genes using the in vitro DNA cleave assay. Analyze GFP expression in the vasa-GFP line to determine if the promoter is active in ovaries and if the RNA is localized to the posterior end of the embryo. Confirm site-specific integration of the CG14427-rtTA M2 construct. Determine if rtTA M2 is active by feeding mothers doxycycline and measuring the level of lethality of female offspring. Make additional germline promoter constructs if the vasa promoter is inactive. e.g. bicoid gene promoter.
Impacts
What was accomplished under these goals?
IMPACT. Spotted wing Drosophila (SWD) (D. suzukii) larvae are significant pests in stone fruits (peach, cherry, and plum) and berries (caneberries, blueberries, and strawberries). D. suzukii is an invasive pest from Asia first discovered in the USA in 2008 and is now widespread. As current insecticide treatments are of limited effectiveness (e.g. weather dependent), non-chemical means for controlling this destructive invasive pest are needed. The New World screwworm (NWS) (Cochliomyia hominivorax) is a devastating pest of warm-blooded animals. One of the greatest achievement's of the USDA in the 20th century was the development of the sterile insect technique (SIT) for the eradication of C. hominivorax from the U.S.A initially and subsequently from Mexico all of Central America. To prevent re-infestation from South America, sterilized flies are currently being constantly released in a "buffer zone" in eastern Panama and along the border with Colombia. While desirable, it would be very expensive and challenging to use SIT to suppress screwworm in countries where it remains endemic (e.g. Brazil, Colombia, Cuba, Jamaica). Cas9-mediated gene drives offer a potentially very efficient genetic mechanism for suppressing populations of insect pests. In theory, a gene drive could effectively suppress insect populations with a single release of 1 male for every 10 fertile males in the region. That is, 100 times fewer males than SIT, which makes a gene drive suppression mechanism economically attractive. However, they pose a challenge for regulators as, by their very nature, they are designed to not be contained. The overall goal of our study is to engineer novel conditional (i.e. with an ON/OFF switch) contained gene drives and evaluate efficacy and safeguards in D. suzukii and C. hominivorax. The drives will target the genes essential for female development or reproduction. This research will provide information for regulators on the use of conditional gene drives to suppress an insect population. With additional engineering, it would be possible to develop strains containing drives that could be suppressed in the laboratory but would drive in the field. Such strains could provide a very efficient mechanism for suppression of these major insect pests. Objective 1. Create conditional female to male transformation and female sterile CRISPR/Cas9 split gene drives in D. suzukii (years 1-2). Estimate 35% complete To build conditional drives requires identification of gene promoters active in the male or female germline (i.e. cells in testes or ovaries that will form mature sperm and eggs respectively). Promoters from genes that make short RNAs such as the U6 genes (U6 RNA is essential for RNA splicing) are needed for expression of guide RNAs (gRNAs) for Cas9. Lastly, to make the system conditional on the addition of doxycycline, the most active form of the reverse tetracycline transactivator (rtTA) in flies needed to be identified. rtTA, a transcription activator, would be used to control expression of Cas9 in the germline. Before assembling the final gene drives we must test the efficacy of guide RNAs (gRNAs) as it is well known that some gRNAs are more efficient than others at directing the Cas9 nuclease to the targeted gene. We previously designed multiple gRNAs and made expression constructs using the Drosophila U6 promoter. We initially planned to evaluate gRNAs by injecting embryos plasmid DNA that would express Cas9 and a specific gRNA. DNA was extracted from the larvae that developed from the injected embryos and analyzed using a real time PCR based DNA melting assay. This assay is reported to be able to detect small deletions at the target site. However, after a number of experiments varying the injection and PCR conditions we were unable to consistently detect specific mutations. The high level of natural DNA polymorphism in the lab strain is likely confounding results from this assay and causing "false positives". Consequently, we switched to an in vitro (i.e. test tube) DNA cleavage assays with purified DNA template, Cas9 protein and gRNA. With this assay we have been able to identify the best gRNA for all of the target genes. To evaluate germline promoters for driving Cas9 expression, the previously generated nanos-Cas9 and vasa-cas9 lines were crossed with a transgenic line that expresses a guide RNA for white. The % of male offspring that develop white eyes provides a measure of Cas9 activity. Three independent crosses were set for each Cas9 line and between 500 and 1000 male offspring were scored for eye color. The three nanos-Cas9 lines evaluated produced 0, 8 and 9% of males with white eye. The three vasa-Cas9 lines gave 2%, 20-80% and 10-50% white-eyed males. Although the nanos-Cas9 lines were less active, the nanos (nos) promoter was more specific for the germline and would be the better choice for a gene drive. Since there had been little previous work with the TET-ON system, we are evaluating three reverse tetracycline trans-activators (rtTAs) that only bind to DNA in the presence of tetracycline. To test activity in Drosophila a construct was made with each rtTA and a GFP gene that would be induced by rTTA. Transgenic lines were made but did not show the expected green fluorescence in ovaries and testes after being fed doxycycline. It is possible that the nos promoter has too low activity for this assay. To address this possibility we made rtTA overexpression constructs. The expectation is that, when fed doxycycline, rtTA overexpression would reduce viability. To improve our SWD transformation efficiency and to target regions in the genome known to be permissive for gene expression, we used Cas9, a gRNA and a long single stranded oligonucleotide template to insert an attP recombination site into a specific location in the D. suzukii genome. After breeding to homozygosity, the strain could be used for inserting gene constructs (eg U6-gRNA) into the specific location using the bacterial phiC31 recombinase. Objective 2. Create conditional female to male transformation CRISPR/Cas9 split gene drives in C. hominivorax (years 1-2). 35% complete The NWS nanos, vasa, bicoid and beta-tubulin germline promoters were identified in the screwworm genome. Gene constructs have been made with the vasa and bicoid promoters. To ensure correct localization of the Cas9 RNA, the 3'UTR was identified for the vasa gene but not nanos. Transgenic Lucilia lines have been made with a vasa promoter-GFP-vasa 3'UTR gene construct. If the promoter is active, ovaries are expected to show green fluorescence. To assay for rtTA function, a transgenic line was made by inserting the CG14427 promoter-rtTA M2 construct into the EF3E line along using phiC31 recombinase. The EF3E line contains an attP recombination site adjacent to an rtTA-inducible female lethal gene. The expectation is that rtTA will be expressed throughout the embryo and will bind to the tetO sequence and activate expression of the lethal gene, causing the female embryos to die. This will only happen if the embryos contain doxycycline that will be fed to mothers. Objective 3. Evaluate the potential for population suppression in cage experiments under permissive and non-permissive conditions (years 2-3). 0% complete. Work on this objective cannot begin until objective 1 or 2 is completed. Objective 4. Evaluate the potential for stopping a drive by releasing flies that are immune to Cas9 (years 2-3). 0% complete. Work on this objective cannot begin until objective 1 or 2 is completed.
Publications
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Progress 09/01/16 to 08/31/17
Outputs
Target Audience:At the third international workshop for regulation of animal biotechnology, Charlottesville, VA, June 26-30, 2017, the PD gave an invited presentation on "Insect biotechnology: current uses and future developments". This included a discussion of how gene drive systems could be used for suppression of insect pest populations. Changes/Problems:It took longer than expected to recruit a suitable postdoctoral fellow. Dr. Bolz started midway through year 1 and spent the first couple of months training on embryo injections and reviewing the literature on spotted wing Drosophila, CRISPR/Cas9 and gene drives. The rtTA TET-ON system has not been widely used in insects. In searching the literature it became apparent that three different sequence variants of rtTA had been developed for mammalian systems. All 3 variants have been made with a New World screwworm codon bias. We propose to evaluate by using previously characterized embryo promoters to drive expression of rtTA in screwworm. What opportunities for training and professional development has the project provided?The postdoctoral fellow, Dr. Kara Bolz, had several years experience working with Drosophila melanogaster but no prior experience in making transgenic flies or with Drosophila suzukii. Dr. Bolz reecived training on how to rear D. suzukii, how to manufacture needles for microinjection, how to microinject Drosophila embryos and how to identify transgenic flies through expression of the fluorescent protein marker gene. How have the results been disseminated to communities of interest?At the Project Director's meeting for the Biotechnology Risk Assessment Grants (BRAG) Program (May 23, 2017), Riverdale, MD, the PD gave a poster presentation on "Development and evaluation of safeguards for conditional suppressive gene drives for spotted wing Drosophila and the New World screwworm ". As noted above, the PD gave a presentation at the "Third international workshop for regulation of animal biotechnology", Charlottesville, VA, (June 26-30, 2017) on "Insect biotechnology: current uses and future developments". What do you plan to do during the next reporting period to accomplish the goals?At this stage the research program is on track. After evaluating gRNA efficiency, conditional gene drive systems will be assembled in D. suzukii as desribed in the report. For New World screwworm, we will proceed with evaluating the performance of rtTA variants for TET-ON conditional expression systems. gRNAs for targeted genes will be evaluated using a similar approach described for D. suzukii.
Impacts
What was accomplished under these goals?
IMPACT. Spotted wing Drosophila (SWD)(D. suzukii) larvae are significant pests in stone fruits (peach, cherry, and plum) and berries (caneberries, blueberries, and strawberries). D. suzukii is an invasive pest from Asia first discovered in the USA in 2008 and is now widespread. As current insecticide treatments are of limited effectiveness (e.g. weather dependent), non-chemical means for controlling this destructive invasive pest are needed. The New World screwworm (Cochliomyia hominivorax) is a devastating pest of warm-blooded animals. Females lay their eggs in open wounds and the hatched larvae eat the animal alive leading to death if untreated. One of the greatest achievement's of the USDA in the 20th century was the development of the sterile insect technique (SIT) for the eradication of C. hominivorax from the U.S.A initially and subsequently from Mexico all of Central America. To prevent re-infestation from South America, sterilized flies are currently being constantly released in a "buffer zone" in Southern Panama and along the border with Colombia. While desirable, it would be very expensive and challenging to use SIT to suppress screwworm in countries where it remains endemic (e.g. Brazil, Colombia, Cuba, Jamaica). Cas9-mediated gene drives offer a potentially very efficient genetic mechanism for suppressing populations of insect pests. In theory, a gene drive could effectively suppress insect populations with a single release of 1 male for every 10 fertile males in the region. That is, 100 times fewer males than SIT, which makes a gene drive suppression mechanism economically attractive. However, they pose a challenge for regulators as, by their very nature, they are designed to not be contained. The overall goal of our study is to engineer novel conditional (i.e. with an ON/OFF switch) contained gene drives and evaluate efficacy and safeguards in D. suzukii and C. hominivorax. The drives will target the genes essential for female development or reproduction. This research will provide information for regulators on the use of conditional gene drives to suppress an insect population. With additional engineering, it would be possible to develop strains containing drives that could be suppressed in the laboratory but would drive in the field. Such strains could provide a very efficient mechanism for suppression of these major insect pests. Objective 1. Conditional split gene drives for SWD. Estimate 25% complete. Note the postdoctoral fellow did not start on this project until midway through year 1. Before assembling the final gene drives we must test the efficacy of guide RNAs (gRNAs) as it is well known that some gRNAs are more efficient than others at directing the Cas9 nuclease to the targeted gene. Consequently, multiple guide RNAs (4-6 per gene) were designed for transformer (a mutation would cause female to male transformation), nudel, and yellow-g (both female sterile when mutated) using a computer program called ChopChop. This program suggests which gRNAs would be the most specific for the targeted gene. Plasmid DNA constructs were made for expression of the gRNAs in SWD embryos. The plasmids use the U6 snRNA gene promoter for expression. Additionally, gRNAs were synthesized using a T7 RNA polymerase system. We are in the process of testing the gRNAs by injecting plasmid DNA and/or synthesized RNA into SWD embryos that express the Cas9 protein. Since there had been little previous work with the TET-ON system, we are evaluating three reverse tetracycline trans-activators (rtTAs) that only bind to DNA in the presence of tetracycline. The three rtTA genes were codon usage optimized for expression in NWS and synthesized by Genscript. For use in SWD, we have cloned the rtTAs with the D. melanogaster nos promoter and SWD nos 3'UTR. The nos promoter is specifically active in the male and female germline, which is essential for gene drive. The nos 3'UTR is required for correctly targeting the RNA to posterior end of the embryo where germ cells will form. Genscript has synthesized the SWD nos promoter, which will also be cloned and compared to the melanogaster promoter. The rtTA gene constructs were combined with a (TetO)7-ZsGreen reporter gene. Once transgenic SWD are obtained with the gene construct, the expectation is that rtTA will be expressed in male and female germline and will bind to the tetO sequence and activate ZsGreen expression and consequently green fluorescence. However, rtTA should only activate ZsGreen in the presence of tetracycline. We are currently verifying the final plasmid DNA clones before proceeding with embryo injections. The promoter/rtTA combination with the best expression of ZsGreen will be used for the Cas9 gene drive construct. That is, we will replace ZsGreen with Cas9. Objective 2. Conditional split gene drives for NWS. Estimate is 25% complete. ?To determine if the TET-ON system can be used in NWS, rtTA genes have been cloned with a screwworm early embryonic promoter. The promoters used are the NWS nullo promoter and Cm CG14427 (secondary screwworm) promoter, which we have previously shown to be active in throughout the embryo. The construct with the CG14427 promoter has been assembled and is awaiting verification. PhiC31 integrase will be used to insert the construct into the genome of an existing (TetO)21-RFPex line of Lucilia (sheep blowfly) to test the strength of the rtTAs. The expectation is that rtTA will be expressed throughout the embryo and will bind to the tetO sequence and activate RFPex expression, causing the embryos to show red fluorescence. This will only happen if the embryos contain tetracycline. Objectives 3 and 4 are projected to be started near the end of year 2.
Publications
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