Progress 09/01/23 to 08/31/24
Outputs
Target Audience:Regulators. Scientists who regulate GMOs are a target audience. The PD attended the annual PD directors meeting. The PD directors meeting is usually well attended by regulators. In the past, the PD has meet with regulators in Panama who would consider applications for field testing transgenic screwworm. Stakeholders. For the screwworm project, COPEG and USDA-APHIS are important stakeholders. The PD met weekly with technical staff from USDA-APHIS to discuss progress on transgenic screwworm. The PD also participates in monthly calls with USDA-APHIS administrators and provides an update on progress on engineering screwworm for enhanced genetic control. For D. suzukii, the PD met regularly with USDA-APHIS scientists and staff based in Salinas, CA. We are planning large cage field trials of D. suzukii strains to be held on their site starting in the spring of 2025. Berry and cherry growers are important stakeholders as they would benefit from any tools for better control of the pest. The PD does not have an extension appointment but does communicate regularly with extension faculty at NCSU who do work with small fruit growers. In the past the PD has given presentations at national and local meetings that were attended by growers. Scientists. The PD and postdoctoral fellow attended and gave presentations at scientific conferences as described in "how the results were disseminated". Several manuscripts were in preparation from this research for publication in peer-reviewed journals. Graduate and undergraduate students. The PD teaches a class every other fall on "Genetic Pest Management" that is mostly taken by entomology graduate students. The class began mid-August 2024 with 8 graduate students. The PD also taught a 3-week module in a Genetics and Genomics overview class that is taken each fall by graduate students from many disciplines. The module is on genetic pest management but with a particular focus on gene drive. Changes/Problems:No new changes in personnel in the past year. But the project was delayed for the reasons described in a previous report. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?The PD gave presentations at International meetings and at a research symposium in North Carolina. The conferences and talks given were: · The 27th International Congress in Entomology, Kyoto, Japan, August 25-30, 2024. Talk on "Male-only strains for genetic biocontrol of spotted wing Drosophila and the New World screwworm". · First Research Co?ordination Meeting (RCM) on the FAO/IAEA Co-ordinated Research Project "Improvement of Drosophila suzukii Mass-Rearing and Released Methods for SIT Programmes" in Vienna, Austria from July 8-12, 2024. Presentation: "Development and evaluation of Drosophila suzukii auxin-inducible genetic sexing strains". · Nöthiger meeting on the evolution insect sex determination from 2 April - 5 April 2024 at Anacapri, Italy. Talk on: "Homing gene drive in Drosophila suzukii targeting doublesex and an update on sex determination systems in blowflies". · The 2024 University Research Symposium, NC State University, Raleigh, NC, March 18, 2024. Talk on "CRISPR-based gene drives for suppression of insect pests" The PD met weekly with USDA-APHIS stakeholders that are sponsoring related projects on the New World screwworm. In addition, the PD gave monthly presentations on progress on developing genetic systems for screwworm control to senior administrators from USDA-APHIS , USDA-ARS and COPEG. Over the reporting period the presentations have all been virtual. The PD taught a 3-week module on "Genetic Biocontrol using Gene Drive" in the "Genetics and Genomics Survey" class in fall 2023. The class was taken by 15 graduate students that were majoring in a wide range of subjects. The postdoctoral fellow, Dr. Ramyasoma gave two presentations on his research: NC State Genetics and Genomics Academy Flash Talks, Raleigh, NC, August 19, 2024 talk on "Drosophila suzukii Y chromosome-targeted transgenesis and gene expression." 3rd Annual Genetics and Genomics Academy Retreat, NC State University, NC, August 23, 2024: poster on "Drosophila suzukii Y chromosome-targeted transgenesis and gene expression." What do you plan to do during the next reporting period to accomplish the goals?For D. suzukii, to complete assembly of a functional YLE: Test if chromatin insulators can improve Cas9 expression at the Y chromosome site successfully targeted in the past year. Develop additional Y chromosome landing sites by using CRISPR/Cas9 to insert inverted phiC31 recombination sites at locations identified by ATAC-seq as likely open chromatin. Similarly, increase our collection of autosomal landing sites by targeting phiC31 recombination sites to locations identified by ATAC-seq as in an open conformation and in between genes. This will improve our ability to test gene constructs. Develop transgenic lines that express sgRNAs targeting the X-linked haploinsufficient gene wupA Cross sgRNA lines with beta-tubulin Cas9 and collected male offspring. Cross males with wild type females. A strong sex bias with few female offspring would indicate successful editing of the targeted X-linked haploinsufficient gene in the male germline. Assemble a Y-linked editor strain by targeting the Y chromosome with the most efficient Cas9 and sgRNA genes identified through previous experiments. The genes will be flanked with the most effective chromatin insulators and targeted to the location on the Y chromosome that gave the highest level of expression. For C. hominivorax, we will continue testing germline promoters for Cas9 by using a relatively rapid transient expression assay we have developed for testing tissue-specific gene promoters. This involves injecting a line that carries a DsRed fluorescent protein gene that is activated by the tTA transcription factor. Embryos are injected with a plasmid that contains the promoter of interest driving tTA. We used this assay to identify gene promoters active in the salivary gland (Belikoff et al, 2024). The results are obtained in a few days, which is much faster than the 3-4 months it takes to make and evaluate transgenic lines.
Impacts
What was accomplished under these goals?
Objective 1. Create Y-linked gene editor strains in D. suzukii Estimate 75% complete To achieve this objective, we needed to identify unique landing sites on the Y chromosome to target Cas9/sgRNA gene constructs. We also needed gene promoters for expression of Cas9 in the germline and for expression of sgRNAs that are targeting X-linked haploinsufficient genes. For expression of Cas9, we previously reported that constructs had been made with three D. suzukii gene germline promoters with one of two different terminators for a total six Cas9 gene constructs. Each construct contained a ZsGreen fluorescent protein marker gene. At the ends of each construct were phiC31 attB recombination sites. To test the gene constructs at the same location in the genome, we sought to establish a targeted recombination system for germline transformation. At the end of the previous reporting period, germline transformation experiments were underway with a piggyBac vector that contained inverted attP phiC31 sites flanking the sepia eye color gene and a phiC31 gene with a nanos promoter for germline expression. We now report that eight transgenic lines were obtained. Seven of the transgenes were mapped to the X, second or third chromosomes. Fortuitously, one transgene was mapped to the Y chromosome. The precise location of each transgene was determined by splinkerette PCR. The insertion on the Y chromosome was in a region we had previously identified as likely open chromatin by using the ATAC-seq method. The level of phiC31 RNA in testes and ovaries was determined by qRT-PCR. As the lines express the recombinase, germline transformation can be achieved by simply injecting embryos with plasmid DNA that contains inverted attB sites. Recombination mediated by phiC31 recombinase would replace the genes between the attP sites with genes located between the attB sites on the plasmid DNA. Thus, we would obtain lines that contain the Cas9 and ZsGreen genes with loss of the sepia eye color and phiC31 genes. Since we want to obtain a YLE strain, we initially injected embryos from the nos-phiC31 Y chromosome line with each of the six Cas9 constructs. We found that recombination-mediated cassette exchange or RMCE was efficient with stable lines established for each Cas9 construct. The level of Cas9 RNA in testes was measured by qRT-PCR. Cas9 activity was estimated by crossing with lines that express sgRNAs that target the white eye color and yellow body color genes. These assays indicate that Cas9 activity of all lines was low, although some were higher than others. It would appear that low Cas9 line expression is low due to heterochromatic nature of the Y chromosome. To mitigate this effect, we are currently preparing new Cas9 gene constructs flanked by chromatin insulators that have heterochromatin blocking activity. The basis of a YLE strain for population suppression is targeting a Y-linked Cas9 to haploinsufficient X-linked genes in the male germline. Consequently, four gene constructs were made that express two or three sgRNAs targeting the X-linked RpS6 and RpL36 genes. These genes are haploinsufficient in D. melanogaster, a close relative of D. suzukii. The constructs were targeted to a location on the third chromosome using CRISPR/Cas9. The sgRNA lines were each crossed with a nos-Cas9 line that expresses Cas9 in the germline. Male offspring that had one copy of each transgene were collected and crossed with wild type females. Our expectation was that female offspring would die due to inheritance of an X chromosome with loss of function mutations in the RpS6 and RpL36 genes. However, equal numbers of males and females were obtained. From the nos-Cas9/RpS6 male parent, all offspring showed no evidence of mutations in the targeted RpS6 gene. Our current hypothesis is that the mutations were made in the RpS6 gene at too early a stage of spermatogenesis and the defective sperm were eliminated. We plan to repeat this experiment with Cas9 lines driven by a beta-tubulin promoter that is active at a later state of spermatogenesis. From the nos-Cas9/RpL36 male parent, a high percentage offspring had loss-of-function mutations in the RpL36 gene. We conclude that the RpL36 gene is not haploinsufficient in D. suzukii. Consequently, we have made new constructs that express sgRNA targeting the wupA gene, which has recently been shown to be haploinsufficient in D. melanogaster. Objective 2. Create Y-linked gene editor strains in C. hominivorax Estimate 40% complete As for objective one, to achieve this objective we needed to identify unique landing sites on the C. hominivorax Y chromosome to target Cas9. We also needed gene promoters for expression of Cas9 in the germline and for expression of sgRNAs. We have worked with USDA-ARS collaborators to analyze improved gene assemblies of the secondary screwworm Cochliomyia macellaria and primary screwworm C. hominivorax. The combination of PacBio HiFi reads and trio-binning has produced very high quality assemblies, including likely Y-linked scaffolds. We are currently identifying potential Y-linked genes that are expressed in embryos as regions near these active genes could be potential locations for insertion of Cas9 and sgRNA genes. For targeted germline transformation in Lucilia cuprina, two lines have been established with phiC31 recombination sites. Embryos from these lines have been injected with RNA encoding phiC31 recombinase and Cas9 gene constructs containing recombination sites. However, no transgenic have been obtained to date. We plan to continue germline transformation experiments with phiC31 recombinase and also with piggyBac vectors. Objectives 3 and 4. Evaluate the potential for population suppression in cage experiments and evaluate the potential for impacts on non-target populations in cage experiments. Estimate 0% complete Work on these objectives cannot commence until objectives 1 and 2 are completed.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Yadav, A.K., Asokan, R., Yamamoto, A., Patil, A.A. and Scott, M.J. (2024) Expansion of the genetic tool box for manipulation of the global crop pest Drosophila suzukii: Isolation and assessment of eye color mutant strains. Insect Mol. Biol. 33(2): 91-100. https://doi.org/10.1111/imb.12879
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Novas, R., Basika, T., Williamson, M.E., Fresia, P., Menchaca, A. and Scott, M.J. (2023) Identification and functional analysis of Cochliomyia hominivorax U6 gene promoters. Insect Mol. Biol. 32(6): 716-724. https://doi.org/10.1111/imb.12875
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Progress 09/01/22 to 08/31/23
Outputs
Target Audience:see the section on how results have been communicated under "Accomplishments" Changes/Problems:No new changes in personnel in the past year. But the project has been delayed for the reasons described in the previous report. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?The PD gave presentations at International and National meetings and gave an invited presentation in Uruguay on our screwworm research. The conferences and talks given were: The 23rd International Congress of Genetics, Melbourne, Australia, July 16-21 2023. Talk on "Male-only and split homing gene drive strains for genetic biocontrol of agricultural pests". The 65th Livestock Insect Workers Conference, Fredericksburg, Texas, June 11-14, 2023. Talk on "Progress towards improved genetic biocontrol of two livestock pests: primary screwworm and the Australian sheep blow fly". 2023 BRAG PD meeting, virtual, May 23-24, 2023. Talk on "Assessing the Influence of Genetic Background on the Efficacy of Drosophila suzukii Male-only and Gene Drive Strains". The Institut Pasteur de Montevideo, Montevideo, Uruguay, April 27, 2023, invited talk on "Genetic biocontrol of the New World screwworm and spotted wing Drosophila". The PD met weekly with USDA-APHIS stakeholders that are sponsoring related projects on the New World screwworm. In addition, the PD gave monthly presentations on progress on developing genetic systems for screwworm control to senior administrators from USDA-APHIS , USDA-ARS and COPEG. Over the reporting period the presentations have all been virtual. The PD taught a 3-week module on "Genetic Biocontrol using Gene Drive" in the "Genetics and Genomics Survey" class in fall 2022. The class was taken by 15 graduate students that were majoring in a wide range of subjects. The PD also taught a graduate class on "Genetic Pest Management" in fall 2022, which was taken by 8 students. What do you plan to do during the next reporting period to accomplish the goals?Dr Kalindu Ramyasoma now has a year experience working with Drosophila suzukii. He has successfully performed ATAC-seq on three life stages and made several gene constructs for random and targeted transgenesis. He has identified sites to target on the Y chromosome. He has become proficient at collecting and injecting D. suzukii embryos and so I expect he will soon have success making transgenic lines. Over the next year Dr. Ramyasoma plans to: Make transgenic lines with nos-phiC31 transgenes for efficient phiC31-mediated recombination. Make several transgenic with a fluorescent protein gene flanked by inverted attP or attB sites for targeted transgenesis. Make transgenic lines with Cas9 transgenes at the same location in the genome. These will be tested for activity by crossing with existing gRNA strains. Make transgenic lines that express the gRNAs targeting the X-linked haploinsufficient genes. Make gene constructs for targeted transgenesis of the Y chromosome. For C. hominivorax, we will continue testing germline promoters for Cas9 by making transgenic L. cuprina lines that carry the Cas9 transgenes. We will use random (piggyBac) and targeted (phiC31) transgenesis.
Impacts
What was accomplished under these goals?
Objective 1. Create Y-linked gene editor strains in D. suzukii Estimate 45% complete To achieve this objective, we needed to identify unique landing sites on the Y chromosome to target Cas9. We also needed gene promoters for expression of Cas9 in the germline and for expression of sgRNAs. We previously reported on a collaboration with Dr. Simon Baxter at the University of Melbourne, Australia that led to the assembly of a D. suzukii genome from male DNA. Y-linked scaffolds were identified. Eleven putative Y-linked genes were identified by BLAST searches with each of the 16 genes that are Y-linked in D. melanogaster. In addition, Dr. Ramyasoma mapped Illumina reads from RNAseq of RNA from male and female larvae and pupae to the genome. This analysis identified confirmed that most of the D. suzukii Y-linked genes were actively transcribed in male larvae or pupae. Our working model is that Y-linked genes active at the larval stage will have a more open chromatin structure and thus be more easily targeted with our knockin strategy. The ATAC-seq method can be used to identify open chromatin regions. Dr. Ramyasoma performed ATAC-seq with nuclei isolated from mixed sexed embryos and male third instar and pupae. Larvae and pupae were sexed by crossing males from a line with an X-linked ZsGreen gene with wild type females and collecting the non-fluorescent offspring. Several locations on the Y chromosome were identified to be in open chromatin conformation in all three stages. We then selected our top candidates for Cas9 knockin using three criteria; (1) the site was not within or very close to genes, (2) there was at least 500 bp of unique sequence flanking the site, (3) there was high read coverage at all stages showing the site targeted was very accessible. Having identified locations to target we can now proceed with making knockin gene constructs. For expression of Cas9, we previously reported that constructs had been made with three D. suzukii gene promoters that are active in the germline. They were the promoters from nanos (nos), bag-of-marbles (bam) and beta2-tubulin (beta2-tub) genes. The Cas9 used in these constructs had a single nuclear localization sequence or NLS. Subsequently, we found that Cas9 gene with two NLS was more active in D. suzukii. Transgenic lines were madethat express NLS-Cas9-NLS from the D. suzukii nos promoter. These lines were made using apiggyBac transposon vector and have high Cas9 activity. Theywere very effective for homing gene drive(2021 BRAG award). For this project we wanted to compare the Cas9 constructs at the same location using targeted transgenesis. Dr. Ramyasoma prepared six gene constructs two with each germline promoter driving NLS-Cas9-NLS that had either a viral p10 pA terminus or the 3'UTR/pA from the same gene as the promoter (ie nos, bam, beta2-tub pA). The constructs all contained a ZsGreen fluorescent protein gene and were flanked with inverted attB sites for phiC31 recombinase-mediated recombination. We previously reported the generation of a line with inverted attP sites flanking a DsRed fluorescent protein gene. Embryos from this line were injected with plasmid DNA for three of the constructs, nos-Cas9-nos pA (600 embryos), bam-Cas9-bam pA (200 embryos), beta2-tub-Cas9-beta2-tub pA (400 embryos) along with plasmid DNA and RNA that encoded phiC31 recombinase. The adult survivors (38,49 and 26 respectively) were crossed back to wild type. The expectation was that there would be gain of green fluorescence and loss of red fluorescence if there had been successful recombination. However, no recombinants were found. In Drosophila melanogaster, the frequency of phiC31 recombination was significantly improved by using transgenic lines that express phiC31 recombinase in the germline. Consequently, we made piggyBac constructs with a nos-phiC31 gene and injections were underway at the end of the reporting period. Objective 2. Create Y-linked gene editor strains in C. hominivorax Estimate 35% complete As for objective one, to achieve this objective we needed to identify unique landing sites on the C. hominivorax Y chromosome to target Cas9. We also needed gene promoters for expression of Cas9 in the germline and for expression of sgRNAs. In the past reporting period we completed the analysis of the C. hominivorax Y-linked scaffolds. A paper describing the work was published this year (Tandonnet et al). We also completed work on the C. hominivorax U6 gene promoters reported previously and submitted a paper for publication. The manuscript was accepted but not yet published at the end of the reporting period (Novas et al, 2023) To aid in the identification of Y-linked genes in C. hominivorax, we have collaborated with USDA-ARS scientists to assemble the genome for the closely related species C. macellaria. For this genome, we used the trio-binning method. This approach produced a very high quality assembly with much of the Y chromosome on a single scaffold. Encouraged by these results we are collaborating with scientists in Uruguay (who worked on the U6 promoters) to improve the assembly of the C. hominivorax genome. For expression of Cas9, new gene constructs were made with the screwworm nos, bam and beta2-tubulin gene promoters driving Cas9 with 2 NLS. The constructs were cloned into a piggyBac vector and also into a vector with ZsGreen fluorescent protein gene and inverted attP sites for targeted transgenesis. For piggyBac transformation, a new helper plasmid was made with the hyperactive version of the transposase driven by the strong hsp83 gene promoter. Over 1000 embryos have been injected with the piggyBac-Cas9 constructs but to date no transgenic lines have been obtained. Injections with gene constructs carrying inverted attP or attB for targeted transgenesis were underway at the end of the reporting period. Objectives 3 and 4. Evaluate the potential for population suppression in cage experiments and evaluate the potential for impacts on non-target populations in cage experiments. Estimate 0% complete Work on these objectives cannot commence until objectives 1 and 2 are completed.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Yadav, A.K., Butler, C., Yamamoto, A., Patil, A.A., Lloyd, A.L. and Scott, M.J. (2023) CRISPR-Cas9 based split homing gene-drive targeting doublesex for population suppression of the global fruit pest, Drosophila suzukii. PNAS, 120 (25): e2301525120. https://doi.org/10.1073/pnas.2301525120.
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Progress 09/01/21 to 08/31/22
Outputs
Target Audience:see the section on how results have been communicated under "Accomplishments" Changes/Problems:We previously reported that Dr. Anandrao Patil agreed to work on this project following completion of a two-year project on transgenic corn planthoppers. Unfortunately, Dr. Patil decided to move to another in the USA to work on a project more suited to his interests. He left the Scott lab at the end of 2021. However, in the few months he worked on the project he made a number of gene constructs and evaluated several gRNAs using in vitro Cas9 assays. Upon Dr. Patil's departure, the postdoc position was widely advertised. Unfortunately, the quality of applicants in the first round was less than desired. Consequently, the position was re-advertised. For the second round, there were a few outstanding applications. After conducting interviews (via zoom), the position was offered to Dr. Kalindu Ramyasoma. In Sri Lanka, Dr. Ramyasoma successfully made several transgenic Aedes aegypti strains that express microRNAs that should greatly reduce the capacity of the mosquito to transmit dengue virus. Dr. Ramyasoma was the first to make transgenic insects in Sri Lanka. After obtaining his J-1 visa, Dr. Ramyasoma traveled to Raleigh and started in the Scott lab on August 1, 2022. These changes in personnel (Dr. Yadav replaced by Dr Patil was has been replaced by Dr. Ramyasoma) have significantly impacted our ability to progress on this project. However, with Dr. Ramyasoma now in Raleigh we anticipate more rapid progress on achieving our goals over the next year. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?The PD attended the annual BRAG PD meeting in 2022. The PD attended and gave talks at two national and two international conferences on progress on developing screwworm and spotted wing Drosophila strains for genetic biocontrol. The conferences and talks given were: · 2022 Synthetic Biology: Engineering & Design (SEED), Arlington VA., May 2-5, 2022. Invited talk on "Genetic Control of Agricultural Pests". · Nöthiger meeting on insect sex determination from 30 March - 2 April 2022 on the island Schiermonnikoog, the Netherlands. Talk on: "Sex determination, dosage compensation and genetic control of blow flies". · 2022 Genetic Biocontrol Gordon Research Conference in Ventura, CA from June 26 to July 1, 2022. Invited talk on "Male-only Strains for Suppression of New World Screwworm and Spotted Wing Drosophila Populations". The 26th International Congress in Entomology, Helsinki, Finland , July 17-22, 2022. Talk on "Sex determination, dosage compensation and genetic biocontrol". The PD met weekly with USDA-APHIS stakeholders that are sponsoring related projects on the New World screwworm. In addition, the PD gave monthly presentations on progress on developing genetic systems for screwworm control to senior administrators from USDA-APHIS , USDA-ARS and COPEG. Over the reporting period the presentations have all been virtual. The PD taught a 3-week module on "Genetic Biocontrol using Gene Drive" in the "Genetics and Genomics Survey" class in fall 2021. The class was taken by 16 graduate students that were majoring in a wide range of subjects. What do you plan to do during the next reporting period to accomplish the goals?With Dr. Patil's replacement, Dr Kalindu Ramyasoma, joining the lab we anticipate making more rapid progress on achieving the goals. Dr. Ramyasoma will be trained in D. suzukii rearing and embryo microinjection. Since he has prior experience in making transgenic mosquitoes, our expectation is that Dr. Ramyasoma will relatively quickly become proficient in embryo injection with our equipment. Over the next year Dr. Ramyasoma plans to: Identify potential target sites on the Y chromosome by identifying genes that are active in larvae and genes that have an open chromatin conformation. For the latter we will use the ATAC-seq approach. Active genes will be identified by completing the RNAseq analysis begun previously. Evaluate the two NLS Cas9 gene constructs by targeting them to the FL19 location on chromosome 3. The Cas9 lines will be crossed to our existing lines that express gRNAs that target Cas9 to genes that when mutant produce flies with an easily visible phenotype. Cas9-mediated mutagenesis of the white gene will produce offspring with white eyes, instead of the wildtype red eyes. Determine if genes can be targeted to a location near the Y-linked Yp gene. The gene construct was made previously and includes inverted attP sites and a ZsGreen fluorescent protein genes. If successful, phiC31 integrase can be used to insert Cas9 and gRNA genes to this Y-linked location. For C. hominivorax, we will continue testing germline promoters for Cas9. As piggyBac-mediated transgenesis was unsuccessful, we will use Cas9 and HDR to target the genes to specific locations in the genome. The strategy will be similar to that adopted in D. suzukii with gene constructs containing homology arms for HDR-mediated integration, inverted phiC31 attP sites and fluorescent protein marker genes.
Impacts
What was accomplished under these goals?
Objective 1. Create Y-linked gene editor strains in D. suzukii Estimate 35% complete To achieve this objective, we needed to identify unique landing sites on the Y chromosome to target Cas9. We also needed gene promoters for expression of Cas9 in the germline and for expression of sgRNAs. We previously reported on a collaboration with Dr. Simon Baxter at the University of Melbourne, Australia that led to the assembly of a D. suzukii genome from male DNA. The publicly accessible genome assemblies are from females. For our assembly, all of the males used to prepare genomic DNA had the same father. Thus, they all shared the same Y chromosome. Dr Baxter identified sixty-eight putative Y-linked contigs and 13 putative Y-linked genes. In the past year we performed illumina sequencing of RNA from separated male and female larvae and pupae. A strain with an X-linked DsRed fluorescent protein gene was used to sort sexes. The illumina reads are now being aligned to the genome to identify Y-linked genes that are active in larvae. Our working model is that Y-linked genes active at the larval stage will have a more open chromatin structure and thus be more easily targeted with our knockin strategy. For expression of Cas9, we previously reported that constructs had been made with three D. suzukii gene promoters that are active in the germline. They were the promoters from nanos (nos), bag-of-marbles (bam) and beta2-tubulin genes. The Cas9 used in these constructs had a single nuclear localization sequence or NLS. Subsequently, Dr. Anand Patil obtained preliminary evidence that a Cas9 gene with two NLS was more active in corn planthoppers than Cas9 with a single NLS. This was consistent with the higher activity of D. suzukii Cas9 lines made by Dr. Omar Akbari's lab that used the two NLS version of Cas9. Consequently, Dr. Patil made a new construct that used the D. suzukii nos promoter to drive expression of Cas9 with two NLS. The construct also included the nos 3'UTR for localization of Cas9 RNA to the posterior pole of the developing egg, precisely where germ cells form in the embryo. Unfortunately, Dr. Patil left the lab at the end of 2021 (see section on major changes) and was unable to inject the gene construct. However, as the Cas9 gene would also be useful for gene drive (2021 BRAG project), Dr Yadav recently injected this construct and obtained 3 transgenic lines that are being evaluated but appear quite promising. We proposed to target a Cas9 transgene onto the Y chromosome by using CRISPR and homology dependent repair or HDR. The Y chromosome is largely heterochromatic, which may make it difficult to knockin the Cas9 transgene to the selected site. As a first to test of the knockin approach, we injected embryos with Cas9/sgRNA complex plus a plasmid that contained 1 kb homology arms to either side of the Cas9 target site. In between the homology arms were two inverted phiC31 integrase attP sites flanking a GFP fluorescent protein gene. We selected a site near the integration position of the original FL19 transgene as this genome location appears accessible in the germline and gave high levels of transgene expression (Li et al, 2021, Pest Management Science, 77: 4915-4922). A total of 170 embryos were injected with the mixture from which 29 G0 adults developed. Of these 24 were fertile and 4 produced transgenic offspring. The knockin approach thus appears to be very efficient, at least at this site on chromosome 3. The next step will be to inject one of these lines with a plasmid containing inverted attB sites, a DsRed fluorescent protein gene and a source of phiC31 integrase (Likely RNA/plasmid DNA mix). Objective 2. Create Y-linked gene editor strains in C. hominivorax Estimate 30% complete As for objective one, to achieve this objective we needed to identify unique landing sites on the C. hominivorax Y chromosome to target Cas9. We also needed gene promoters for expression of Cas9 in the germline and for expression of sgRNAs. We previously reported that our collaborator, Dr. Philippos Papathanos (Hebrew University, Israel) had used the chromosome quotient or CQ analysis to identify potential Y-linked contigs/genes. We reported that nine of the putative 36 Y-linked genes were tested by PCR but found to be autosomal and not on the Y chromosome. It appeared that the initial CQ analysis had a high false positive discovery rate. We have been collaborating with Dr. Sophie Tandonnet (University of Sao Paolo, Brazil) on annotation of a new HiC assembly of the C. hominivorax genome. D. Tandonnet performed a new more stringent CQ analysis. Ten potential Y-linked scaffolds were identified. Four were selected for testing by PCR with male and female genomic DNA. All four scaffolds have Y-linked material, although one scaffold appears to be about 50% autosomal (first 50%). Thus, this analysis has identified several potential landing sites for Cas9 genes. For expression of Cas9, we previously reported that gene constructs have been made with the screwworm nos, bam and beta2-tubulin gene promoters driving the tetracycline transactivator or tTA gene. The advantage of taking this approach was that the tissue-specific expression pattern of tTA can be detected by crossing to an established strain that carries a tTA-activated DsRed gene. To test the activity of the gene promoters, embryos from the Australian sheep blowfly, Lucilia cuprina were injected with the plasmids and a source of piggyBac transposase. As we are unable to work with C. hominivorax in Raleigh, we have previously successfully tested several C. hominivorax gene promoters in L. cuprina, which is a related blowfly. Embryos were injected with the three germline promoter constructs in a piggyBac transformation vector. The embryos were co-injected with a mix of plasmid DNA and RNA as sources of piggyBac transposase. From these injections more than 100 G0 adults developed for each gene construct. We would have anticipated two to five independent transgenic lines for each construct based on our previous experience. Unfortunately, no transgenic lines were obtained. It is unclear why these injection experiments failed. It is does not appear to be because of mutations as the complete DNA sequences for all plasmids was determined using illumine DNA sequencing. Investigations to why piggyBac-mediated transgenesis failed are ingoing but in the interim we have proceeded with developing transgenic lines using a Cas9 knockin strategy. We previously developed a rapid in vivo assay to evaluate C. hominivorax U6 gene promoters. In brief, embryos from a transgenic L. cuprina line that carries a DsRed gene were injected with a mix of plasmid DNA and Cas9 protein. The plasmid contains a U6 promoter driving the expression of a gRNA that targets Cas9 to the DsRed gene. Surviving G0 first instar larvae are collected, DNA isolated, a fragment of the DsRed gene amplified by PCR and the amplicon sequenced using illumina technology. Of the six C. hominivorax U6 promoters evaluated, three produced a higher percentage of mutations (insertions/ deletions or INDELS) than control injections without Cas9 protein. The mutagenesis frequency ranged from 150 to 650 INDELS per 100,000 reads. The control activity was about 50 reads per 100,000. The most active U6 gene promoter has been selected for evaluation with gRNAs that target X-linked genes. Objectives 3 and 4. Evaluate the potential for population suppression in cage experiments and evaluate the potential for impacts on non-target populations in cage experiments. Estimate 0% complete Work on these objectives cannot commence until objectives 1 and 2 are completed.
Publications
|
Progress 09/01/20 to 08/31/21
Outputs
Target Audience:Regulators. Scientists who regulate GMOs are a target audience. The PD will attend the annual PD directors meeting and present either a poster or give a talk if invited. The PD directors meeting is usually well attended by regulators. In the past, the PD has meet with regulators in Panama who would consider applications for field testing transgenic screwworm. If requested by COPEG, the PD will meet again with Panamanian regulators. Stakeholders. For the screwworm project, COPEG and USDA-APHIS are important stakeholders. The PD meets weekly with technical staff from USDA-APHIS to discuss progress on transgenic screwworm. The PD also participates in monthly calls with USDA-APHIS administrators and provides an update on progress on engineering screwworm for enhanced genetic control. For D. suzukii, berry and cherry growers are important stakeholders as they would benefit from any tools for better control of the pest. The PD does not have an extension appointment but does communicate regularly with extension faculty at NCSU who do work with small fruit growers. In the past the PD has given presentations at national and local meetings that were attended by growers and hopefully will do so again (post pandemic). Scientists. The PD will attend and give presentations at national and international scientific conferences. If invited, the PD will give presentations at other Universities. Results from this research will be published in peer-reviewed journals. Graduate and undergraduate students. The PD teaches a class every other fall on "Genetic Pest Management" that is mostly taken by entomology graduate students. The class teaches all aspects of genetic pest management, including Y-linked approaches such as in this project. The PD also teaches a 3-week module in a Genetics and Genomics overview class that is taken each fall by students from many disciplines. The module is on genetic pest management but with a particular focus on gene drive. Changes/Problems:As access to the laboratory was restricted due to the Pandemic for much of the reporting period, we delayed hiring a full-time postdoctoral fellow. Rather the reported progress was achieved through collaborations (Y chromosome genes) and by existing personnel such as Dr. Amarish Yadav working part-time on this project. We have now identified a postdoctoral fellow to work on this project. Dr. Anandrao Patil has been in the Scott lab for over two years working on making conditional lethal genes in the corn planthopper. Dr. Patil is a very experienced molecular biologist. After some training on how to rear D. suzukii and microinject embryos, I expect Dr. Patil to drive this project to a successful completion. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?The PD attended the annual BRAG PD meeting in 2021 and gave a brief presentation. The PD gave an invited talk via zoom to the Department of Entomology, Cornell University on September 21, 2020. The title of the presentation was "Development of genetic systems for control of insect pests" and included progress on developing screwworm and spotted wing Drosophila strains for genetic control. The PD met weekly with USDA-APHIS stakeholders that are sponsoring related projects on the New World screwworm. In addition, the PD gave monthly presentations on progress on developing genetic systems for screwworm control to senior administrators from USDA-APHIS , USDA-ARS and COPEG. Over the reporting period the presentations have all been virtual. The PD taught the "Genetic Pest Management" class in the fall semester 2020. The class was taken by six graduate and two undergraduate students. The students were mostly majoring in entomology or genetics. The PD taught a 3-week module on gene drive in the "Genetics and Genomics Survey" class in fall 2020. The class was taken by 15 graduate students that were majoring in a wide range of subjects. What do you plan to do during the next reporting period to accomplish the goals?For D. suzukii we are now well placed to make gene constructs targeting the identified unique landing sites on the Y chromosome. sgRNAs for targeted X-linked genes will be tested in vitro and U6 promoter constructs made with the most efficient sgRNAs. Embryos will be injected with the Cas9 gene constructs and transgenic lines identified through expression of fluorescent protein marker genes. For C. hominivorax, we will continue testing and evaluating germline promoters for Cas9 and U6 promoters for sgRNAs. sgRNAs for selected X-linked genes will be evaluated using in vitro and in vivo assays. We will continue testing for Y chromosome linkage of putative Y chromosome genes. Lastly, Cas9/sgRNA gene constructs will be made targeting locations on the Y chromosome. As targeting specific locations in the genome has not yet been accomplished, we will also make gene constructs for targeting autosomal locations that prior research has identified are optimal for gene expression and male fitness.
Impacts
What was accomplished under these goals?
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 achievements 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). As the screwworm outbreak in the Florida Keys in 2016 clearly demonstrated, the USA remains at risk of re-invasion of this major livestock pest. Genetic approaches for efficient suppression of C. hominivorax in countries where it is endemic and for future control of outbreak(s) in the USA are needed. The advent of CRISPR/Cas9 technology has revolutionized many areas of genetics including genetic systems for potential pest control. One very promising strategy is to insert Cas9 and sgRNA genes onto the Y chromosome. This is sometimes referred to as the Y-linked editors strategy. Y-linked gene editors offer a potentially very efficient genetic mechanism for controlling populations of insect pests compared to the sterile insect technique (SIT) and release of insects with conditional dominant female lethal genes (fsRIDL). Modeling indicates that Y-linked gene editor strains would have significantly less impact on non-target populations with which there is some gene flow compared to homing gene drive strains. However, to date there is no experimental support of these claims. We proposed to build Y-linked editor strains targeting X-linked genes required for viability of XX females. We will then evaluate the potential of the strains for population suppression and also any impact on non-target populations. The information gained from these experiments will be valuable for regulators in assessing a future field release of Y-linked editor strains. Objective 1. Create Y-linked gene editor strains in D. suzukii Estimate 25% complete To achieve this objective we needed to identify unique landing sites on the Y chromosome to target Cas9. We also needed gene promoters for expression of Cas9 in the germline and for expression of sgRNAs. We previously identified two Y-linked genes in D. suzukii. To increase the potential number of unique landing sites we have collaborated with Dr. Simon Baxter at the University of Melbourne, Australia. We supplied DNA from male flies that shared the same father and thus had identical Y chromosomes. PacBio HiFi DNA sequencing was then performed with the DNA. Dr. Baxter used the output to assemble a male genome of D. suzukii using Flye. All of the currently available D. suzukii genomes were assembled from female DNA. The male genome assembly size is 429 Mb across 3791 contigs. To identify Y-linked contigs we performed Illumina sequencing on male and female DNA. Dr. Baxter aligned the Illumina reads to the contigs. Sixty eight putative Y-linked contigs were identified as many more reads from the male DNA mapped to these contigs than reads from female DNA. To date 13 putative Y-linked genes have been identified in these contigs, including the two genes we previously identified. This analysis has provided many more potential Cas9 landing sites. For expression of Cas9, we identified the nos, bam and beta2-tubulin gene promoters in the genome assembly. The promoters were synthesized with restriction sites for cloning. Cas9 gene constructs have been made with germline promoters. One construct has been made for targeting Cas9 to the region near the Y-linked Pp1-Y2 gene. The most efficient U6 promoters, U6-1 and U6-3 were identified in the genome assembly. The promoters were synthesized with restriction sites for golden gate cloning. The next step will be to evaluate sgRNAs that target X-linked genes Objective 2. Create Y-linked gene editor strains in C. hominivorax Estimate 20% complete As for objective one, to achieve this objective we needed to identify unique landing sites on the Y chromosome to target Cas9. We also needed gene promoters for expression of Cas9 in the germline and for expression of sgRNAs. We collaborated with Dr. Philippos Papathanos (Hebrew University, Israel) to identify potential Y-linked genes. We performed Illumina DNA sequencing on DNA from male and female C. hominivorax. Dr. Papathanos then used the chromosome quotient or CQ method to identify potential Y-linked genes from the genome we previously assembled and annotated. When the Illumina reads are mapped back to the genome, many more reads from the male DNA will map to potential Y-linked genes. This analysis identified 36 Y-linked genes, including one of the two genes we had previously confirmed was on the Y chromosome. Nine genes were tested for Y-linkage by PCR of male and female DNA. None of the genes appear to be on the Y chromosome. The CQ analysis appears to have a high false positive discovery rate. This analysis will continue but at this stage we do not have any additional unique landing sites other than the previously identified Y-linked genes. For expression of Cas9, we identified the nos, bam and beta2-tubulin gene promoters in the genome assembly. The promoters were synthesized with restriction sites for cloning. To confirm male germline expression we have initially synthesized gene constructs with the promoters driving the tetracycline transactivator or tTA gene. An advantage of taking this approach is that the tissue-specific expression pattern of tTA can be detected by crossing to an established strain that carries a tTA-activated DsRed gene. If the promoters are found to be active and germline-specific, Cas9 gene constructs will then be made with the promoters. Five U6 genes were identified in the genome. The promoters were synthesized with restriction sites for golden gate cloning. We have developed a relatively quick in vivo assay for U6 promoter activity. sgRNAs that efficiently target Cas9 protein to the DsRed, ZsGreen an endogenous yellow genes were identified by injecting pre-cellular Lucilia cuprina embryos with Cas9/sgRNA pre-assembled complexes. Gene constructs have been made with each U6 promoter and a selected sgRNA for the DsRed gene. The activity of the gene constructs will be assessed by injection of the plasmid DNA with Cas9 protein. Objectives 3 and 4. Evaluate the potential for population suppression in cage experiments and evaluate the potential for impacts on non-target populations in cage experiments. Estimate 0% complete Work on these objectives cannot commence until objectives 1 and 2 are completed.
Publications
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