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. Stakeholders. For this project, USDA-APHIS is an important stakeholder. 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. In an effort to reach a wider community interested in gene drive we regularly participate in the weekly meetings of the NC State University Genetic Engineering and Society center. The meetings alternate between in person and virtual. The in person meetings are well attended by faculty and students from the social and biological sciences. The virtual meetings are open more widely and often include people from other states and other countries. Dr Yadav has given presentations to this group on his gene drive work in D. suzukii (see dissemination section). Scientists. The PD, postdoctoral fellow (Yadav) and PhD student (Tarrand) 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:The one major problem we have had this year was a severe mite infestation in the ACL2 containment room that we use for gene drive experiments. Consequently, we were unable to complete the large cage population suppression experiment for objective one. All cages were shutdown, frozen and then autoclaved. The ACL2 room was cleaned with dilute bleach and wiped down with 95% ethanol. We are now re-establishing populations in the large cages. The cage sleeves have been thoroughly cleaned and treated with a moldex solution to inhibit mold growth. The suppression experiment will begin once stable populations are established in the cages. What opportunities for training and professional development has the project provided?Dr Yadav attended the Southeastern NIH Biotechnology Training Grant Career Development Workshop held at Duke University in June 2024. He also attended several meetings and gave talks or presented posters as listed under how results were disseminated. Ariel Tarrand is mostly supported through an NIH training grant awarded to the Biotechnology program. A requirement of the program is that students complete a 3-month internship at a biotech company. In the summer of 2024, Ms Tarrand was an intern at Biocentis, the only biotech company in the world focusing on developing gene drive strains of insect pests. Biocentis is located in Italy. Ms Tarrand also attended the Gordon Research Conference on Genetic Biocontrol in Barcelona, Spain in May 2024. She received the award for best poster presentation at the meeting. 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 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. Dr Yadav gave presentations on his research at several meetings listed below: · The 27th International Congress in Entomology, Kyoto, Japan, August 25-30, 2024. Talk on "CRISPR/Cas9-based split homing gene drive for genetic suppression of the global crop pest, Drosophila suzukii". · Southeastern NIH Biotechnology Training Grant Career Development Workshop held on June 13thand June 14th ,2024 at Duke University, Durham, USA. Presented a poster entitled "CRISPR/Cas9-based split homing gene drive for population control of the crop pest, Drosophila suzukii" · The Allied Genetics Conference (TAGC24), Washington DC, March 6-10, 2024. Presented poster entitled as "CRISPR/Cas9-based homing gene drive for population control of the crop pest, Drosophila suzukii". · Annual meeting of The Entomological Society of America (ESA) November 5-8, 2023 National Harbor, Maryland. Presented a virtual poster entitled 'CRISPR/Cas9-based homing gene drive for control of the crop pest, Drosophila suzukii'. Colloquium organized by theGenetic Engineering and Society Center, NC State University (NCSU), USA, October 3, 2023. Delivered a talk on 'CRISPR/Cas9-based gene-drive to suppress agricultural pests'. https://ges.research.ncsu.edu/event/colloquium-2023-10-03/ Ariel Tarrand gave presentations at the following meetings: · Gordon Research Conference on Genetic Biocontrol, Barcelona, Spain, May 19-24, 2024. Presented a poster titled "Genetic background has little effect on suppression gene drive efficiency in spotted wing Drosophila". Received first prize. · ESA Southeastern Branch Meeting in Augusta, GA, March 18, 2024. Gave a talk titled "Impact of Genetic Background on Gene Drive Homing Rate inDrosophila suzukii". · Integrative Symposium of NC State Training Grants, Raleigh, NC, Feb 9, 2024. Presented a poster titled "Life Finds a Way - Or Does It? Testing the Limits of Gene Drive Resistance in Diverse Populations of Spotted-Wing Drosophila" What do you plan to do during the next reporting period to accomplish the goals?We are well placed to complete the objectives in the next reporting period. For each objective, the remaining small and large cage population suppression experiments will be performed using our established procedures. For objective 1, we will complete evaluation of drive efficiency for the lines that express two or more gRNAs targeting doublesex
Impacts
What was accomplished under these goals?
Objective 1. Estimate 85% complete We previously reported very high gene drive efficiency (94-100% inheritance), with a single gRNA targeting the female exon of the doublesex gene. The initial drive strains were dominant female sterile. By modifying the drive to include an optimal splice acceptor, only homozygous females were sterile (i.e. recessive sterility). Modeling indicated that both dominant sterile and recessive sterile split drives could suppress populations at ratios as low as 1:4 (gene drive male: wild type male) each generation. In the current reporting period, we performed discrete generation population suppression experiments in small cages. Each strain and control were tested with four replicate cages. As predicted from modeling, a 1:4 release ratio with males carrying the dominant female gene drive was sufficient to eradicate the cage populations in nine generations. However, for the experiments with the recessive sterile gene drive males it was necessary to double the release ratio midway through the experiment. Eradication was achieved after 14 generations. Follow-up fitness experiments found that the recessive gene drive males were outcompeted by the wild type males if an X-linked Cas9 was used. With an autosomal Cas9 gene the drive males were fully competitive. We then began larger cage continuous population suppression experiments but these had to be abandoned before they were completed due to a severe mite infestation (see problems/changes section). Homing gene drives can fail if the Cas9-mediated double-stranded DNA break is repaired by the NHEJ pathway and the mutation generated is resistant to Cas9 cleavage and encodes a functional protein. We previously reported that simple in-frame 3 bp or 6 bp deletion at the gRNA cut site did not produce a functional DOUBLESEX protein and thus would not stop drive. Nevertheless, this does not rule out other mutations such as simple insertions that may produce a functional protein and stop drive. As the potential for resistance can be reduced if more than one gRNA is expressed targeting the female doublesex exon, we made and evaluated homing gene drives that express two or more gRNAs. Multiple gRNAs can be produced by using a different U6 promoter to express each gRNA or by using tRNA processing to excise the gRNAs from a single transcript. Dr Yadav took both approaches, making gene constructs that express two gRNAs using two U6 promoters or tRNA processing. Gene drive lines were obtained as before by using CRISPR/Cas9 to insert the drive construct into the female doublesex exon. Since this is a split drive system, homing gene drive males and females were obtained with the two U6 promoter construct by crossing to the existing Cas9 lines, both autosomal and X-linked. The homing gene drive males and females then have one copy of a Cas9 gene (on the X or second chromosome) and one copy of the disrupted doublesex gene with a multi-gRNA gene and DsRed marker gene. The drive males and females were then each crossed with wild type and the offspring scored for inheritance of the red fluorescent protein gene that was inserted into the doublesex gene. If there is no homing drive, 50% of the offspring will show red fluorescence. If homing is perfect, then 100% of the offspring will show red fluorescence. With homing gene drive males, about 85% of the offspring showed red fluorescence. The efficiency was lower than with a single gRNA but still highly significant. However, with homing gene drive females, only about 60% of the offspring showed red fluorescence. We can't explain why there is such a difference in homing rates between the sexes. With the two gRNAs, the Cas9 cut sites were 50 bp apart. Perhaps homology-dependent repair is more sensitive to distance between the cut sites in females. At the end of the reporting period crosses were underway with the gene drive strains that should produce the same two gRNAs but from a single transcript after tRNA processing. We have, however, evaluated two drive constructs supplied by Dr. Champer that each produce three gRNAs from a single transcript. From the crosses with homing gene drive males and females to wild type only about 55-60% of the offspring inherited a red fluorescent protein gene. This is much lower efficiency than we previously obtained with a single gRNA. To determine if the low drive efficiency was due to a high rate of deletion/insertion mutations in the doublesex exon due to NHEJ repair, DNA will be isolated from non-drive offspring and the doublesex female exon amplified and sequenced. Objective 2. Estimate 90% complete For the genetic background experiments with the fsRIDL male-only FL19 strains, we previously reported significant variation in the level of dominant female killing among strains collected from different states. Female lethality ranged from highly effective 99% (Maryland [MD], North Carolina [NC] and Washington [WA]) to as low as 71-80% (Michigan [MI], Oregon [OR], California [CA]). There was no clear east-west divide as might have been predicted from whole genome sequencing of flies caught at various locations across the USA. In the current reporting period, we performed discrete generation population suppression experiments with the NC, OR and CA strains. Each control and test cage population received 20 pairs at the start of the experiment. The test populations also received 60 (3:1 ratio) FL19 males obtained by rearing the FL19 strain on diet without tetracycline. As previously published, in the highly effective NC genetic background, the small cage populations were eradicated within four generations. Surprisingly, in the less effective OR and CA genetic backgrounds, the populations were also eradicated within four generations. Thus, we conclude that genetic background differences don't impair the ability of FL19 fsRIDL males to suppress populations. However, to confirm this conclusion we have begun to test the ability of FL19 males to suppress continuous generation populations in large lab cages. Objective 3. Estimate 70% complete Since homing gene drive was very efficient with the single gRNA line in our standard NC genetic background, we have been measuring drive efficiency in different genetic backgrounds. For these experiments, it was advantageous to use an autosomal Cas9 line for the crossing scheme so that sons would inherit the Cas9 line from fathers. We chose the C25A2 line that was shown to have very high activity. Males carrying one copy of Cas9 and one copy of the doublesex drive allele were crossed with wild type females from the strains collected from different states. We previously reported that in the NC genetic background, average gene drive efficiency was 97% (four replicate experiments). Drive efficiency is defined as % offspring that inherit the doublesex drive allele that carries the DsRed gene. Without homing, 50% of the offspring should inherit the doublesex drive allele. The experiment has now been performed with females from CA, OR, Minnesota (MN), MI and NJ strains. Gene drive efficiency was high in all genetic backgrounds with a slight variation from 92% (NJ, CA, OR, MI) to 96% (MN). The drive efficiency was not significantly different among the different genetic backgrounds. Thus, genetic background does not appear to impact homing gene drive efficiency in D. suzukii. We will next test the ability of gene drive males to suppress small cage populations with strains from other states such as CA and OR.
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
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Progress 09/01/22 to 08/31/23
Outputs
Target Audience:The PD gave a talk at the annual BRAG PD meeting and gave talks on our gene drive research as listed under accomplishments Changes/Problems:No major problems to report. Dr Amarish Yadav has agreed to stay in the lab and work on this project for the next year. Ariel Tarrand has begun the second year of her doctoral studies and will continue to investigate gene drive efficiency in genetic backgrounds. She is supported by a traineeship from the NC State biotechnology program. What opportunities for training and professional development has the project provided?The postdoctoral fellow, Dr. Amarish Yadav, gave an invited talk at the retreat for the Genetics and Genomics Academy (GGA) on August 25. The GGA is cross-college academy at NC State. Dr Yadav was awarded the "Paper-of-the-Year" in recognition of his paper entitled "CRISPR-Cas9 based split homing gene-drive targeting doublesex for population suppression of the global fruit pest, Drosophila suzukii ", which was published in PNAS in 2023. Dr Yadav has been invited to present his work at the International Congress of Entomology in Kyoto in August 2024. 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 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". 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 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?We are well placed to meet most of the objectives in the next reporting period. For objective 1, lines that express two or more gRNAs targeting dsx will be established and their drive efficiency measured by crossing with a Cas9 line. We will begin population suppression experiments with small cages. For objective two, a population suppression experiment will be undertaken with one of the wild type strains that had a high level of female survival. For objective 3, we will test drive efficiency in different genetic backgrounds following the same protocol established for the California strain. After completing these experiments, we will perform a population suppression experiment with a strain that a relatively low drive efficiency and compare with our North Carolina strain.
Impacts
What was accomplished under these goals?
Objective 1. Estimate 75% complete As we reported previously, drive efficiency for the initial system tested was much lower than reported for Anopheles mosquitoes. To improve drive efficiency, four new Cas9 lines were made that used the D. suzukii nos promoter to drive expression of Cas9 with two NLS. Two lines were X-linked (27A1, C25A2) and two were autosomal (C25A2, C25A3). To assess Cas9 activity, each line was crossed with lines that express gRNAs for the yellow and white genes. Loss-of-function mutations in yellow and white produce flies with a yellow body and white eye, respectively. These can be easily distinguished from wild type flies that have a grey body and red eyes. All of the Cas9 lines were active, with the 27A1 X-linked line producing the most yellow body mutants (65%) and the C25A2 autosomal line producing the most white eyed flies (100%). Consequently, the 27A1 and C25A2 lines were selected for homing gene drive experiments. To measure gene drive, homozygous Cas9 virgin females were crossed with hemizygous males from a dsx disrupted strain. These strains carry a red fluorescent protein gene and dsx gRNA gene inserted into the female exon of dsx. As described in the previous report, we previously generated dsx dominant female sterile lines (3A3) and recessive female sterile lines (1C2 and 23AI[SA]). Crosses were performed with each dsx mutant strain. The offspring were crossed with wild type flies to measure homing efficiency. If there was no homing, 50% of the offspring would be expected to inherit the DsRed gene inserted into dsx. If drive was fully efficient, 100% of the offspring would show red fluorescence. For a female derived Cas9 allele from the X-linked 27A1 Cas9 line, inheritance was 97% for 3A3, 98% for 1C2 and 98% for 23AI[SA]. That is, drive efficiency was almost perfect, a significant improvement over our initial experiments with Cas9 lines carrying a single NLS. Homing gene drives can fail if the Cas9-mediated double-stranded DNA break is repaired by the NHEJ pathway and the mutation generated is resistant to Cas9 cleavage and encodes a functional protein. To address the potential for resistance to drive, we first performed a series of in vitro experiments with Cas9 protein, gRNA and DNA fragments carrying 1 bp mutations at several locations and larger 3 bp or 6 bp deletions that were in-frame. The 1 bp mutations were all efficiently cut by Cas9. However, the 3 bp and 6 bp deletions were completely resistant to cleavage by Cas9. To determine if these mutations could provide functional resistance, Dr. Yadav used long single-stranded oligonucleotides and Cas9/gRNA complex to generate dsx mutant strains carrying a 3 or 6 bp deletion. Genetic testing showed that these mutant alleles did not encode a functional DSX protein. Consequently, small in-frame deletions generations by the NHEJ pathway should not provide resistance to drive. The work was submitted for publication and published in the reporting period (Yadav et al, 2023). Having developed a very efficient gene drive system that may not break down due to functional resistance arising in the population, we could now proceed with testing the ability of released drive males to suppress a cage population of the local North CarolinaD. suzukiistrain. The work had begun at the end of the reporting period. We could also now proceed with objective 3. Lastly, as the potential for resistance can be reduced if more than one gRNA is expressed targeting the female dsx exon, Dr Yadav made gene constructs that express two gRNAs. We also began a collaboration with Dr. Jackson Champer, who is also interested in developing gene drives that target dsx. He provided three constructs, that each express three gRNAs targeting dsx. Microinjections with these new drive constructs had begun at the end of the reporting period. Objective 2. Estimate 85% complete For the genetic background experiments, we obtained strains established with flies caught in three east coast (NJ, MD, MI) and three west coast states (WA, OR, CA). Twenty FL19 males were crossed with 20 virgin females from each strain and the male and female offspring counted. Up to 10 replicate experiments were performed for each strain. With our NC strain we obtained 59 females and 1493 males. Thus we calculate female lethality was 96%. For NJ, MD and MI female lethality was 93% (1673 total), 99.5% (743 total) and 71% (590 total), respectively. For the WA, OR and CA strains, female lethality was 99.5% (690 total), 83% (681 total) and 80% (881 total), respectively. Since genetic analyses had shown that the east coast and west coast populations were distinct and likely derived from flies that originated in different locations in Asia, we anticipated that we may see an east coast/west coast divide with regard to effectiveness of the female-killing system. However, this was not observed with strains from each coast having high female lethality (NJ, MD, WA) or significant female survival (MI, OR, CA). We next will test the ability of the FL19 strain to suppress a population with significant female survival such as the CA strain. Objective 3. Estimate 25% complete Since homing gene drive was very efficient with the new Cas9 lines we have begun to test the effectiveness in different genetic backgrounds. For these experiments, it was advantageous to use an autosomal Cas9 line for the crossing scheme so that sons would inherit the Cas9 line from fathers. We chose the C25A2 line that was shown to have very high activity (objective 1). We first assessed drive efficiency in our North Carolina strain, which serves as a control for these experiments. Males carrying one copy of Cas9 and one copy of the dsx drive allele were crossed with NC wild type females. From four replicates, the drive efficiency was 98%, 99%, 93% and 97% with an average of 97%. Drive efficiency is defined as % offspring that inherit the dsx drive allele that carries the DsRed gene. Without homing, 50% of the offspring should inherit the dsx drive allele. We next crossed males with one copy of Cas9 and one copy of the dsx drive allele with virgin females from a California strain. Male offspring that inherit one copy of Cas9 and one copy of the dsx drive were collected. The genetic background of the males would be a 50/50 mix of North Carolina and California. To assess drive efficiency in their germline, the males were crossed with virgin California females. From five replicates, the drive efficiency was 94%, 96%, 87%, 89% and 99% with an average of 93%. Drive efficiency was more variable than in the NC background and averaged slightly lower efficiency. However, the difference in drive efficiency was not statistically significant. We next plan to repeat these experiments with other wild type strains.
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:The PD attended the annual BRAG PD meeting and gave talks at National and International conferences (listed under accomplishments) Changes/Problems:No major problems to report. Dr Amarish Yadav agreed to stay in the lab and work on this project. He previously worked on homing gene drive and Y-linked Cas9 systems in Drosophila suzukii on other BRAG grants (2016-33522-25625, 2020-33522-32317). With his considerable experience working with Drosophila suzukii, Dr Yadav made very good progress in the first year of the project. Dr Aki Yamamoto is assisting Dr Yadav with the gene drive experiments and leading the work with the FL19 strains. The rearrangement in the two-gRNA construct that prevented expression of one gRNA was unfortunate but not viewed as major problem as alternative approaches exist. It has taken us much longer than anticipated to establish continuous populations of Drosophila suzukii in large cages in the NCSU phytotron. The advantage of using the phytotron is that the growth chambers have controlled temperature, humidity and light. However, we are making progress and, if necessary, we can go back to the smaller cage continuous populations described previously (Li et al, 2021). What opportunities for training and professional development has the project provided?The postdoctoral fellow, Dr. Amarish Yadav, attended the 2022 Genetic Biocontrol Gordon Research Conference in Ventura, CA from June 26 to July 1, 2022 and presented a poster on "CRISPR-Cas9 based split homing gene-drive targeting doublesex for population suppression of the global fruit pest, Drosophila suzukii" 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 meetings and one international conference on progress on developing 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". · 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". What do you plan to do during the next reporting period to accomplish the goals?We are well placed to meet the objectives in the next reporting period. The two gRNA expression construct will either be modified to reduce the chance of rearrangement or we will use the self-cleaving tRNA strategy to express two gRNAs as described in the project narrative. Continuous population suppression experiments will either be performed as previously described (Li et al 2021) or in the larger cages in the NCSU phytotron, if cages can be stabilized.
Impacts
What was accomplished under these goals?
Objective 1. Estimate 30% complete As we reported in the project narrative, drive efficiency for the initial system tested was much lower than reported for Anopheles mosquitoes. For population suppression it is desirable if homing drive efficiency is as high as possible. Thus, much of our efforts in the first year of the project have been directed at improving homing efficiency. Three of the four initial drive lines showed dominant female sterility apparently due to production of the male splice variant. It is not clear why in the fourth line, 1C2, hemizygous females were fertile. As population suppression is more efficient if drive occurs in both sexes, we first worked on redesigning the homing construct to ensure that drive females did not make the male splice variant. The homing construct was modified by the addition of an optimal splice acceptor. Additionally, the sequence following the splice acceptor site encoded a degron motif to destabilize any protein made by dsx transcripts. Four independent dsx knockin lines were obtained and were confirmed to have correctly inserted within the dsx female exon. In contrast to the first drive system, in all four modified lines the hemizygous females were fertile. Thus, drive could occur in both sexes. To measure gene drive, homozygous Cas9 virgin females were crossed with hemizygous males from the dsx disrupted strain 23AI1. The offspring were crossed with wild type flies to measure homing efficiency. If there was no homing, 50% of the offspring would be expected to inherit the DsRed gene inserted into dsx. If drive was fully efficient, 100% of the offspring would show red fluorescence. Homing drive inheritance in 23AI1was higher than most of the original lines with 66% of the offspring showing red fluorescence. However, drive was slightly lower than the 1C2 line (71%). Having solved the issue of dominant female sterility, gene constructs were made to express two gRNAs targeting the dsx female exon. Each construct contained the optimal splice acceptor site and used the D. suzukii U6-1 and U6-3 gene promoters to express the gRNAs. One construct carried a DsRed fluorescent protein marker gene and the other a ZsGreen fluorescent protein gene. The initial Cas9 lines made in our lab carried a ZsGreen marker gene whereas those made by Omar Akbari's lab (UC San Diego) have a DsRed marker gene. Thus, by making the gRNA constructs with red or green markers, we could evaluate homing efficiency with the Cas9 lines from both labs. In total, one red and two green two gRNA expression lines were obtained. While molecular analyses confirmed correct integration within the dsx gene, we also detected a minor rearrangement at the U6-3 promoter. Unfortunately, this mutation would prevent expression of the second gRNA. We are currently investigating what might have caused the instability. We proceeded with performing homing assays as the gRNA expressed from the U6-1 gene promoter had not been previously tested. Drive inheritance of 55% with our Cas9 line nos-36X were less than obtained previously with the 1C2 and 23AI1 lines. It thus appears that homing drive is not as efficient with the new gRNA. Crosses with the Akbari lab BiC-Cas9 line are underway. The Cas9 used in our initial constructs had a single nuclear localization sequence or NLS. Subsequently, we obtained preliminary evidence that a Cas9 gene with two NLS was more active in corn planthoppers than Cas9 with a single NLS. Consequently, we made a new construct that used the D. suzukii nos promoter to drive expression of Cas9 with two NLS. Four independent transgenic lines were obtained by piggyBac-mediated germline transformation. The new Cas9 lines are currently being initially evaluated but appear quite promising. Objective 2. Estimate 70% complete We have made excellent progress on this objective in the first year. As described in the project narrative, we had previously obtained lines with two copies of the female lethal gene construct, FL19, on chromosome 3 and four X-linked lines with a single FL19 gene. One of the four X-linked lines, #7, showed a high level of dominant female lethality when reared on diet without tetracycline. As two copies of FL19 should produce a higher level of female lethality, recombinant lines were obtained with two copies of FL19 on the X chromosome. These were #7 with #77 (7+77) and #7 with #79 (7+79). We developed a relatively rapid single generation assay to assess the potential of new lines to suppress populations. In this assay, transgenic males are mixed with wild type males and wild type virgin females. The ratio of transgenic males to wild type males was 3:1 or 5:1. The female/male ratio of the offspring provided an indication of the competitiveness of the transgenic males. A highly competitive strain would produce a low female to male ratio as most of female offspring would inherit the dominant female lethal gene. For reference, the original single copy FL19 strain led to female/male ratios of 0.62 (3:1) and 0.38 (5:1). The lines with two X-linked FL19 genes appear to be more competitive than the original single copy line. The female/male ratios were 0.2 (3:1) and 0.4 (5:1) for the 7+77 strain and 0.4 (3:1) and 0.3 (5:1) for the 7+79 strain. In contrast, the lines with two closely linked copies of FL19 on chromosome 3 were less competitive than the original single copy line. The female/male ratios with a 5:1 release were 0.7 for the #36 strain and 0.9 for the #8 strain. We next performed discrete generation population suppression assays as previously described (Li et al, 2021, Pest Management Science, 77, 4915). Repeated releases of a 3:1 excess of the original single copy FL19 males led to population extinction after 10 generations. A similar experiment with a 3:1 excess of the two-copy X-linked strains 7+79 and 7+77 led to population extinction after four and three generations, respectively. Repeated releases of a 5:1 excess of the two-copy autosomal strain #36 required 13 generations to drive the populations to extinction. The time to extinction is consistent with the results from the single generation competitiveness assay described above. Given the promise of the two copy X-linked lines we plan to select one of them and test their effectiveness in different genetic backgrounds. For the genetic background experiments, we obtained strains established with flies caught in three east coast (NJ, MD, MI) and three west coast states (WA, OR, CA). Twenty FL19 males were crossed with 20 virgin females from each strain and the male and female offspring counted. Up to 10 replicate experiments will be performed for each strain. Initial results show a wide variation with no females obtained in some backgrounds (WA, MD) and significant female survival in other backgrounds (25% for CA and 30% for MI). Objective 3. Estimate 10% complete Work on this objective will not commence until gene drive efficiency has been optimized (objective one). In preparation for these experiments, one of the original Cas9 lines, nos-36X, has been back-crossed for six generations into each of the different genetic backgrounds.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2022
Citation:
Yadav, A.K., Butler, C., Lloyd, A.L. and Scott, M.J. (2022) CRISPR-Cas9 based split homing gene-drive targeting doublesex for population suppression of the global fruit pest, Drosophila suzukii. PNAS, under review
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