MATERNAL SPECIFIC MUTATIONS CAUSING LOSS OF PRIMODIAL GERM CELL TO PRODUCE STERILE FISH POPULATIONS WITH IMPROVED CULTURE PERFORMANCE.

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1017360
• Grant No.: 2019-67030-29002
• Cumulative Award Amt.: $100,000.00
• Proposal No.: 2017-09036
• Project Start Date: Nov 15, 2018
• Project End Date: Nov 14, 2020
• Grant Year: 2019
• Program Code: [A1801]- Exploratory: Exploratory Research

Recipient Organization
CENTER FOR AQUACULTURE TECHNOLOGIES, INC.
8395 CAMINO SANTA FE STE E
SAN DIEGO,CA 92121

Performing Department
(N/A)

Non Technical Summary
Facing diminishing yields from wild fisheries, global food supplies will have to rely more on the fish farming industry to fulfill an ever-increasing public demand for seafood. A significant limitation of finfish aquaculture is caused by the early sexual maturation of commercial species, which results in billions of dollars in lost productivity and downgraded product quality every year. In addition, farmed fish can escape and negatively impact aquatic ecosystems. To address these economic and environmental hindrances, various reproductive containment solutions have been proposed, but none to date has proved fully effective, or has been widely adopted by the industry.Here, we propose to investigate the effect of precise genetic alterations designed to disrupt the formation of primordial germ cells (PGCs), the progenitors of the gametes. Without PGCs, embryos develop as sterile and sexually immature adults. This approach should overcome the limitations of current sterilization strategies and hopefully provide the quantum leap this industry urgently needs.Our principal objective is to interrogate the function of at least six genes expressed in oocytes and whose products are believed to be necessary for PGC development in fish embryos. We aim to identify those genes exerting maternal effects and that have no other essential biological function. Female fish carrying such edited genes should produce embryos depleted of PGCs. If successful, we will develop methodologies to ensure that new generations of these "grandchildless" tilapia females can be produced. We will also study the performance of their sterile progeny in grow-out trials, measuring important production traits that will be compared to half sibling but fertile counterparts.In summary, we propose to test in tilapia a new sterilization strategy that can be transferred to multiple species and could have a major positive impact on the aquaculture industry. However, the proposed research does carry significant risks inherent to a reverse genetic approach applied to a complex genome. As such, we believe our proposal fits the criteria set forth in the Exploratory Research Program Area Priority.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
301	0810	1050	70%
302	0810	1020	10%
301	0810	1080	10%
303	0810	1081	10%

Knowledge Area
303 - Genetic Improvement of Animals; 302 - Nutrient Utilization in Animals; 301 - Reproductive Performance of Animals;

Subject Of Investigation
0810 - Finfish;

Field Of Science
1081 - Breeding; 1080 - Genetics; 1020 - Physiology; 1050 - Developmental biology;

Keywords

fish aquaculture

primordial germ cells

sterilization

targeted genome editing

tilapia

Goals / Objectives
Objective 1. Evaluate the sterility phenotype associated with partial or complete loss-of-function of six candidate genes in Tilapia (Years 1-2). In this objective, we will produce and evaluate lines of tilapia carrying mosaic mutations in genes we believe are essential for primordial germ cell (PGC, ie. progenitor of the gametes) development. CAT scientists have previously created stable transgenic lines of tilapia expressing the Green Fluorescent Protein (GFP) in PGCs. The GFP-labeled cells will be a crucial tool to evaluate the effect of our mutations. Thus, mutants will be created in tilapia lines containing the GFP marker. We will produce and evaluate at least 3 mosaic females mutated for each gene of interest. If such females carry newly induced maternal-effect mutations, they should be fertile and easily identifiable by scoring for PGC reduction in their embryo progeny. Mutations in genes participating in additional essential developmental processes or essential for germ line development in adults will be eliminated early in our screen from associated F0 lethality or sterility.Objective 2 Establish lines, characterize their phenotypes and demonstrate PGC rescue (Years 1-2). For further phenotypic characterization, it is essential to generate identical mutations in every cell of the animal. This can only be achieved after germline transmission of mutations present in the injected founder fish, which is genetically mosaic. In this objective, we will propagate mutation(s) in gene(s) that confer a maternal-effect sterile phenotype. To this end, we will use F0 mutated males and cross these males with wild type females carrying the GFP transgene. This progeny is fertile given that the maternal effect gene products are provided by the wild type mother. These F1 progeny will be screened to locate progeny carrying the desired mutation. F1 progeny carrying the same mutation of interest will then be crossed to produce F2 progeny. This F2 progeny will be raised to adulthood, sexed and genotyped to identify homozygous mutant. Homozygous mutant females carrying the GFP transgene should produce PGC depleted progeny that will grow into sterile or sub-fertile adult. Sterility will be studied at the morphological cellular and molecular level in 3 and 5-month-old F3 progenies. In contrast to females, homozygous mutant males should produce progeny with a normal PGC count.Objective 3. Quantify production traits and growth rate of sterile populations (Years 2). In Objective 3, we will test the hypothesis that induced sterility can provide increased culture performance by preventing loss of energy to gonad development and sexual differentiation. To test the potential benefit of sterility, we will evaluate the line with the most complete PGC ablation effect. We will perform tank grow-out trials to compare growth performance, and food conversion efficiency between sterile and control groups.

Project Methods
Objective 1. Evaluate the sterility phenotype associated with partial or complete loss-of-function of six candidate genes in Tilapia (Years 1-2). We have identified tilapia orthologues of six selected genes in silico from genomic databases. These select sequences will be targeted following microinjection of engineered nucleases into one cell stage embryos. To enhance the frequency of generating null mutations in the gene of interest, we will target 2 separate exons simultaneously. Alongside the gene of interest, we will target a pigment gene to serve as a mutagenesis selection marker. All mutants will be created in tilapia lines containing the GFP transgene. We expect that injection of 200 embryos will produce 20-60 embryos carrying loss of function mutations. Five of these embryos will be quantitatively assayed for genome modifications by PCR fragment analysis. Furthermore, we will only raise batches of embryos in which mutations were produced at the one or two cell stage, ie. detection of 2 or 4 mutant alleles per targeted loci by fragment analysis assay. Mutants will be screened for morphological malformations, developmental delays and sex differentiation. If the mutated fish develop normally, fertility of 3 males and 3 females will be assessed at 4 and 6 months respectively by crossing them with tilapia lines expressing GFP in PGCs. For each cross, 30 F1 progeny will be genotyped and an additional 20 will be analyzed by fluorescent microscopy. Since these lines express GFP selectively in PGCs, labelled-PGCs can be counted at 4 dpf when all PGCs have completed their migration to the genital ridges. The mean total PGC numbers will be statistically compared across F1 progenies using an unpaired t test. If the engineered mutations function as hypothesized, we expect F1 embryos produced from F0 females to have reduced or absent GFP-PGC counts. Likewise, if the mutations are indeed maternal-effect specific, we expect F0 males to produce F1 progeny with a normal PGC counts (~35+/- 5 PGCs/embryo).Objective 2 Establish lines, characterize their phenotypes and demonstrate PGC rescue (Years 1-2). To select F1 hemizygous (outcrossed with WT fish of different genetic backgrounds) and F2 homozygous lines, we will use QPCR melt analysis (MA) on amplicons spanning the targeted genomic regions. Because each heterozygous lesion produces a characteristic melt curve, it will be possible to regroup and breed F1 progeny carrying the same indels. To fully characterize the indels, we will sequence the PCR products from F1 individuals. Confirmation of sterility at the molecular, cellular and morphological level.For each locus targeted, F3 embryos from F2 homozygous mutant males and females crossed with WT broodstock (n=30/group), will be produced and raised to 2-3 months of age. Gonads from 10 juveniles will be dissected and RNA/cDNA will be screened by QPCR using vasa, a germ cell specific gene. Q-PCRs for each sample will be performed in triplicate and the level of vasa expression will be normalized to a set of host house-keeping genes (b-actin and ef1α). We expect no expression of vasa in sterile fish. At 5 months of age, we expect sterile males to have translucid testes and sterile females to yield a string-like ovary.Objective 3. Quantify production traits and growth rate of sterile populations (Years 2). To generate 3 half sibling groups for these trials, embryos from 3 WT males crossed with 3 F2 homozygous mutant females (sterile groups) and 3 WT females (fertile groups) will be reared separately using established hatchery procedures. At ~1 month of age, tilapia progeny (n=100/group) will be weighed, pit-tagged and held together in 3x300-Liters tanks in a recirculating culture system maintained at 27ºC. All fish will be fed twice daily, to satiation, using a commercially prepared grow-out diet. Each fish will be individually weighed and measured at 4-week intervals over a 24-week period. At the end of the experiment, fish will be sacrificed, sexed, the mean total fish length, weight, filet yield and growth curves will be statistically compared using an unpaired t test.

Progress 11/15/19 to 11/14/20

Outputs
Target Audience:Our target audience includes students, research scientists, and other members of the scientific community, as well as aquaculture producers, and government risk assessor and decision-makers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided training and professional development opportunities for Valerie Williams, PhD, Melissa Hoffman, MS, and Spencer Herbert, BS, who contributed to the molecular and gene editing studies (synthesis of engineered nucleases, microinjection of embryos, analysis of genotypes and phenotypes). The project further provided training on fish husbandry to three individuals with part-time positions, including one undergrad and one BS student. How have the results been disseminated to communities of interest?Our results were presented in conference-talk (Aquaculture America 2020); seminar-talk (Institute of Marine and Environmental Technology-University System of Maryland); in poster and printed abstract (Aquaculture America 2020). Our results were further printed in professional journal (World Aquaculture magazine vol.51, #2 2020) and published in our patent application available online (Publication number: WO/2020/018877, publication date: 23.01.2020, "A method of generating sterile progeny"). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Here, we describe how precise genetic changes can improve production traits in aquaculture. We show how the targeted disruption of specific genes causes females to mass produce sexually delayed population of progeny. This approach offers a simple and practical solution to deal with problems caused by precocious maturation. We further demonstrate the benefit of sterilization to enhance fish growth performance in culture. Objective 2: We raised Igf2bp3-/- tilapia to sexual maturation stage and dissected their gonads. We found immature ovaries arrested at the previtellogenic stage and partially translucid testes with very low sperm count. Altogether, these results indicate that Igf2bp3 contributes to gametogenesis in tilapia. In contrast homozygous female for a Ptbp1a mutant allele are fertile and produce PGC-depleted embryos (Mean number of 15 PGCs/embryo vs.40 PGCs/control embryo). Thus, Igf2bp3 belongs to a group of genes (TIA1, TIAR, ElavL1, Nos3 3'UTR), whose complete or partial inactivation produce no obvious phenotype to adulthood. However, embryos derived from mutant mothers for these genes had, on average, between 65% and 95% of PGC number reduction. In contrast to females, male homozygous mutants for these genes produced progeny with a normal PGC count, indicating no paternal or zygotic effect of these mutations. We raised PGC-depleted progeny from TIA1, TIAR, ElavL1, Nos3 3'UTR and Ptbp1a mutant females to adulthood and analyzed their gonads for size and alterations. We found a positive correlation between the level of PGC ablation and the severity of the sterility phenotype. Embryos with low PGC count developed into sexually delayed male and female with small size testis and ovaries at 6 months of age. Embryos with no visible PGC developed into sterile fish. Sterile males displayed a pair of translucid tube-like testes and sterile females a pair of string like ovaries. Histological sections revealed that these gonads had only somatic gonadal cells. As expected, expression of vasa, a germ cell specific gene marker, was not detectable by Q-PCR in the gonad from sterile fish. Objective 3: We performed tank grow-out trials to compare the growth of full sibling tilapia with or without a functional gonad (GCF: Germ Cell Free). Fish were pit-tagged and held together in 3x300-Liters tanks in a recirculating culture system maintained at 27ºC and fed twice daily, to satiation, using a commercially prepared grow-out diet. Total Body Weight measurements were made at month intervals over 6 months from 2 to 8 months of age. A statistical analysis of body weight gain and specific growth rates provided evidence of significant positive effect of sterility on growth. Monthly average of daily body weight gain indicated that GCF tilapia grew 12% faster than their maturing siblings starting around the time of puberty. Our data are consistent with expectation that sterility improves the growth rate and food conversion ratio, as energy is not lost to gamete development and active mating behavior.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Sterility in Aquaculture - Advances, Performance, Impacts: Gene editing to induce sterility in fish farming Xavier Lauth, J. Buchanan, T. Umazume, M. Hoffman, V. Williams, M. Michelato Kawakami and E. Hidalgo World Aquaculture magazine vol.51 #2 June 2020
• Type: Websites Status: Published Year Published: 2020 Citation: Poster abstract: MAKING PRESCISE GENETIC CHANGES IN THE TILAPIA GENOME Takeshi Umazume , Melissa Hoffman, Valerie Williams, John Buchanan, Xavier Lauth https://www.was.org/Meeting/Program/PaperDetail/156814
• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Virtual seminar to the Institute of Marine and Environmental Technology (IMET) Title: Editing genes for sterility in fish farming May 27.
• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Oral presentation: "Gene editing to induce sterility in fish farming" X.Lauth. National Aquaculture Association special producer session Aquaculture America Conference 2020

Progress 11/15/18 to 10/15/20

Outputs
Target Audience:Our target audience includes students, research scientists, and other members of the scientific community, as well as aquaculture producers, and government risk assessor and decision-makers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided training and professional development opportunities for Takeshi Umazume, BS, Valerie Williams, PhD, Melissa Hoffman, MS, and Spencer Herbert, BS, who contributed to the molecular and gene editing studies (synthesis of engineered nucleases, microinjection of embryos, analysis of genotypes and phenotypes). The project further provided training on fish husbandry to three individuals with part-time positions, including one undergrad and one BS student. How have the results been disseminated to communities of interest?Our results were presented in conference-talk (Aquaculture America 2020); seminar-talk (Institute of Marine and Environmental Technology-University System of Maryland); in poster and printed abstract (Aquaculture America 2020). Our results were further printed in professional journal (World Aquaculture magazine vol.51, #2 2020) and published in our patent application available online (Publication number: WO/2020/018877, publication date: 23.01.2020, "A method of generating sterile progeny"). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Aquaculture currently produces nearly half of all fish for human consumption. Facing diminishing yields from wild fisheries, global food supplies will have to rely more heavily on the fish farming industry to fulfill an ever-increasing public demand for seafood. Thus, it is critical to further develop and expand aquaculture operations while reducing their impact on the environment and improving culture performance. In this study, we developed and investigated new lines of tilapia designed to reduce production cost and alleviate environmental concerns associated with farm escapees. We disrupted the function of six evolutionary conserved genes and studied their contribution to fish reproduction. We found that females for five edited lines produced embryos depleted of primordial germ cells (PGCs: progenitor of the gametes) that grew into a sexually delayed population, maturing at 7-10 months of age as oppose to 4 -6 months of age. We further observed that one female line produced 10% of sterile progeny. We evidenced significant positive effect on growth in tank grow-out trials starting around the time of puberty, with sterile fish having 12% faster growth rates, higher Body Weight Gain (574 g vs 510 g at 8 months of age) and specific growth rates (2.05 vs 1.98) compared to their fertile siblings. While containment is not yet 100% effective, our results establish proof of principle that maternal sterilization is possible. Our solution addresses the problem associated with early sexual maturation, which affects productivity (energy lost to gamete production), fish health and flesh quality, and which causes hundreds of millions of dollars of economic losses globally. We believe this technology can be further improved and is transferrable to any fish species. Objective 1. We generated F0 lines of tilapia carrying indels in six different genes presumed to participate in germ cell development. Furthermore, we targeted a sequence motif in the 3'UTR of nanos3 that we hypothesized is involved in the spatio-temporal regulation of the corresponding mRNA in oocyte and early embryos. We did not observe significant differences in viability or visible gross developmental abnormalities between the F0 edited groups and controls and all F0 mutant groups developed with a normal sex ratio. Next, we investigated the maternal effect of the mutations to determine if they altered the PGC development pathways. For this, 2-4 sibling F0 females in each edited group were bred with wild-type males and their embryo progeny was analyzed under fluorescent microscopy to score PGC numbers. Different F0 females in each KO group produced embryos with varied PGC ablation levels likely due to the mosaicism of sequence outcomes at the target sites. We measured PGC reduction levels ranging from 65% to 80% (averaging 14 to 8 PGCs/embryo), depending on the gene targeted. In contrast, all F0 mutant males crossed with transgenic females (Zpc5:eGFP:nanos 3'UTR) produced embryos with normal PGC count (averaging 40 PGCs/embryo) indicating that the mutations had no paternal or zygotic effect. PGC depleted progeny from F0 mutant females were raised to adulthood and their gonads were analyzed for size and alterations. We found atrophic ovaries as well as partially translucid germ cell depleted testis consistent with the severe PGC loss in embryos. In contrast, progeny from F0 mutant males developed normal sized gonads. Objective 2. We generated F2 progeny carrying the desired loss of function mutation by breeding F1 hemizygous mutants and recovered all anticipated genotypes at the expected Mendelian frequencies with no obvious phenotypes through adulthood. One of the six Knockout (KO) lines (Elavl2-/-) only produced agametic male and female indicating that this gene is essential for the maintenance of the germ line stem cells in adults. Another KO line (Igf2bp3-/-) produced sterile female and subfertile male. For all other edited lines (TIA1-/-, TIAR-/-, Elavl1-/- and Ptbp1a-/-), we crossed sexually matured homozygous mutant females with WT males and analyzed the embryos progeny. We observed strong PGC reduction in the progeny of homozygous females, with PGC ablation levels ranging from 65% to 90% (averaging 15 to 4 PGCs/embryo) depending on the specific gene targeted. We further analyzed the progeny from females lacking a regulatory motif in the 3'UTR region of nanos3 and scored a PGC ablation level approaching 95%. These mutant females had the most severe maternal sterilization phenotype producing 20% of embryos with no visible PGCs while all remaining embryos showed less than 5 PGCs. We raised these embryos to adulthood and found a positive correlation between the level of PGCs ablation and the severity of the sterility phenotype. Embryos with low PGC counts developed into sexually delayed male and female with small and immature testis and ovaries at 6-9 months of age. Embryos with no visible PGCs developed into sterile fish with translucid tube-like testes and string like ovaries.The maternal effect sterilization strategy described here does not require a treatment to reverse sterility and comprises the steps of 1) breeding a fertile hemizygous mutant male and female fish, 2) selecting a female progenitor that is homozygous by genotypic selection, and 3) breeding the homozygous female progenitor to produce the commercial population of sexually delayed/sterile progeny. Objective 3. We performed tank grow-out trials to compare the growth of full sibling tilapia with or without a functional gonad (GCF: Germ Cell Free). Fish were pit-tagged and held together in 3x300-Liters tanks in a recirculating culture system maintained at 27ºC and fed twice daily, to satiation, using a commercially prepared grow-out diet. Total Body Weight measurements were made at month intervals over 6 months from 2 to 8 months of age. A statistical analysis of body weight gain and specific growth rates provided evidence of significant positive effect of sterility on growth. Monthly average of daily body weight gain indicated that GCF tilapia grew 12% faster than their maturing siblings starting around the time of puberty. Our data are consistent with expectation that sterility improves the growth rate and food conversion ratio, as energy is not lost to gamete development and mating behavior.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Sterility in Aquaculture - Advances, Performance, Impacts: Gene editing to induce sterility in fish farming Xavier Lauth, J. Buchanan, T. Umazume, M. Hoffman, V. Williams, M. Michelato Kawakami and E. Hidalgo World Aquaculture magazine vol.51 #2 June 2020
• Type: Websites Status: Published Year Published: 2020 Citation: Poster abstract: MAKING PRESCISE GENETIC CHANGES IN THE TILAPIA GENOME Takeshi Umazume , Melissa Hoffman, Valerie Williams, John Buchanan, Xavier Lauth https://www.was.org/Meeting/Program/PaperDetail/156814
• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Virtual seminar to the Institute of Marine and Environmental Technology (IMET) Title: Editing genes for sterility in fish farming May 27. https://imet.usmd.edu/event/virtual-seminar-dr-xavier-lauth-director-innovation-center-aquaculture-technologies
• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Oral presentation: "Gene editing to induce sterility in fish farming" X.Lauth. National Aquaculture Association special producer session Aquaculture America Conference 2020

Progress 11/15/18 to 11/14/19

Outputs
Target Audience: Nothing Reported Changes/Problems:The project is advancing according to plan. What opportunities for training and professional development has the project provided?The project has provided training and professional development opportunities for two individuals. Valerie Williams, PhD and Melissa Hoffman, MS, to undertake molecular work and learn the techniques to inactivate gene function in tilapia, which include the design and synthesis of engineered nucleases, the microinjection of embryos, the genotyping of mutants and analysis of mutant phenotypes. The project further provided training on fish husbandry for one undergraduate student. How have the results been disseminated to communities of interest?Talks and poster presentations are scheduled for the coming year, including an oral presentation at the Aquaculture America 2020 conference. What do you plan to do during the next reporting period to accomplish the goals?We intend to score the PGC number in the progeny of mutant females in lines that have not yet reached sexual maturity. We further intend to demonstrate the advantage of being sexually delayed or sterile by performing grow-out trials.

Impacts
What was accomplished under these goals? Objective 1. We generated F0 lines of tilapia carrying indels in six different genes presumed to participate in germ cell development. Furthermore, we targeted a sequence motif in the 3'UTR of nanos3 that we hypothesized is involved in the spatio-temporal regulation of the corresponding mRNA in oocyte and early embryos. We did not observe significant differences in viability or visible gross developmental abnormalities between the F0 edited groups and controls and all F0 mutant groups developed with a normal sex ratio. Next, we investigated the maternal effect of the mutations to determine if they altered the PGC development pathways. For this, 2-4 sibling F0 females in each edited group were bred with wild-type males and their embryo progeny was analyzed under fluorescent microscopy to score PGC numbers. Different F0 females in each KO group produced embryos with varied PGC ablation levels likely due to the mosaicism of sequence outcomes at the target sites. We measured PGC reduction levels ranging from 65% to 80% (averaging 14 to 8 PGCs/embryo), depending on the gene targeted. In contrast, all F0 mutant males crossed with females Tg(Zpc5:eGFP:nanos 3'UTR) produced embryos with normal PGC count (averaging 40 PGCs/embryo). PGC depleted progeny from F0 mutant females were raised to adulthood and their gonads were analyzed for size and alterations. We found atrophic ovaries as well as partially translucid germ cell depleted testis consistent with the severe PGC loss in embryos. In contrast, progeny from F0 mutant males developed normal sized gonads. Objective 2. We generated F2 progeny carrying the desired loss of function mutation by breeding F1 hemizygous mutants and recovered all anticipated genotypes at the expected Mendelian frequencies with no obvious phenotypes through adulthood. One of the six KO lines developed into sterile male and female. To measure the full strength of the maternal effect sterility phenotypes, we crossed sexually matured homozygous mutant females with WT males and analyzed the embryos progeny. We observed strong PGC reduction in the progeny of homozygous females for three of the six genes targeted, with PGC ablation levels ranging from 75 to 90% (averaging 10 to 4 PGCs/embryos). In the remaining two mutant lines, females have not yet reached sexual maturity. We further analyzed the progeny from females lacking the nanos3 3'UTR motif and scored a PGC ablation level exceeding 90%.

Publications