Source: CENTER FOR AQUACULTURE TECHNOLOGIES, INC. submitted to NRP
STERILE MONOSEX POPULATIONS FOR GENETIC CONTAINMENT OF GENETICALLY ENGINEERED FISH.
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1017150
Grant No.
2018-33522-28745
Cumulative Award Amt.
$249,838.00
Proposal No.
2018-02682
Multistate No.
(N/A)
Project Start Date
Sep 1, 2018
Project End Date
Aug 31, 2022
Grant Year
2018
Program Code
[HX]- Biotechnology Risk Assessment
Recipient Organization
CENTER FOR AQUACULTURE TECHNOLOGIES, INC.
8395 CAMINO SANTA FE STE E
SAN DIEGO,CA 92121
Performing Department
(N/A)
Non Technical Summary
Multiple species of fish have been genetically engineered (GE) to incorporate advantageous traits for aquaculture or to produce valuable pharmaceutical proteins. Unfortunately, widespread adoption of these GE fish is hampered by concerns over their accidental release to natural ecosystems. Failproof sterilization techniques would be invaluable for minimizing the impact of GE fish lines. Sterile fish have been produced by triploidization, transgenic approaches, and a variety of transient gene silencing methodologies. However, these approaches often remain technically difficult, costly and insufficiently robust or scalable to be used in commercial settings. To discover novel and effective means of sterilizing fish, we propose to investigate induced mutations across a combination of genes necessary for spermiogenesis and steroid hormone synthesis; resulting in male sterility and masculinization respectively. More specifically, double mutant combinations of genes should produce all-male sterile populations. Likewise, double mutant combinations of genes responsible for maleness and folliculogenesis should result in sterile all-female populations. Thus, our proposed project will generate and characterize 14 different lines of tilapia carrying null mutations in two genetic pathways simultaneously, one governing sex differentiation and the other sex/gamete competency. We will also develop strategies to generate broodstock capable of propagating these mutant lines, through use of sex-reversal and transplantation approaches aimed respectively at rescuing the missing gender and restoring gamete production. Finally, we will test the performance of these lines in grow-out trials. If successful, this project will enable safe and effective utilization of GE fish in a wide range of commercial applications. Thus, our project aligns well with USDA BRAG program area #1: "Management Practices to Minimize Environmental Risk of GE Organisms".
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
3033714108050%
3013714102020%
3053714108130%
Knowledge Area
303 - Genetic Improvement of Animals; 305 - Animal Physiological Processes; 301 - Reproductive Performance of Animals;

Subject Of Investigation
3714 - Tilapia;

Field Of Science
1081 - Breeding; 1080 - Genetics; 1020 - Physiology;
Goals / Objectives
Objective 1. Evaluate the sterility phenotype of spermiogenesis deficient all-male tilapia (Years 1-2). In this objective, we will produce and evaluate between 6 and 10 lines of tilapia carrying loss of function mutations in up to two genes essential for female sex development and up to five genes governing spermiogenesis. To engineer male sterility, we previously evaluated the effect of null mutations in a gene controlling an important branch point in steroid hormone synthesis, regulating both androgen and estrogen production. Null mutations in this gene cause fish to develop as male, which remain fertile, but exhibit delayed sexual maturation and reduced sperm counts. We also previously investigated the possibility of blocking spermiogenesis and identified five genes causing subfertility in males (reduced sperm count, motility and morphological deformities), while mutant females remain fully fertile. Thus, in this first objective we will establish lines of tilapia carrying double homozygous mutations to interrogate the effect of simultaneously impairing spermiogenesis and steroid hormone synthesis. We will than test the hypothesis that these double mutants will develop as sterile male populations.Objective 2. Evaluate two genes targeting male differentiation in conjunction with two other genes controlling oogenesis to produce a sterile all-female population (Years 1-3). In Objective 2, we will first produce all-female tilapia populations, by generating null mutations in two genes known in other species of fish to cause genetically programmed males to sex reverse into females. To further engineer sterility in females, we will target four genes that should be essential for vitellogenesis. We will determine whether their loss of function trigger arrest of ovarian development. Next, we will breed these mutants to generate eight lines of tilapia deficient in both masculinization and vitellogenesis. We hypothesize that females in these lines will have small ovaries, with follicles arrested at the previtellogenic stage or yield production of abnormally small non-functional eggs. Thus, we expect that females carrying the newly induced mutations will be sterile.Objective 3. Develop methodology to rescue male and female sterility, and ensure propagation of the lines, and evaluate their performance (Years 2-3). In Objective 3, we will first evaluate approaches to rescue maternal and paternal KO lines and to generate viable broodstock with particular emphasis on germ cell transplantations.In our male sterile KO lines, we hypothesize that the mutants will develop a testicular microenvironment, where spermiogenesis is impaired due to Sertoli-specific defects. Consequently, we expect that transplantation of spermatogonial stem cells from the male knockout infertile donors to a permissive wild type testicular environment, will restore sperm functionality and fertility (allowing for propagation of KO genotypes).Likewise, sterility in mutant female should result from defect in somatic cells (Theca and liver cells). Thus, we hypothesize that oocytes carrying null alleles of these genes should retain their intrinsic capacity to proliferate and differentiate, ensuring that oogonial stem cells from a sterile female mutant donor can re-populate the ovaries and differentiate into functional eggs upon transplantation into a WT/permissive recipient. Thus, we believe that recipient males or females can produce gametes that carry the donor genotypes.Once methodologies to rescue male and female sterility are in place we will investigate the potential benefit of sterility (i.e. growth performance, food conversion etc.) in tank grow-out trials.
Project Methods
Objective 1. Evaluate the sterility phenotype of spermiogenesis deficient all-male tilapia (Years 1-2).In objective 1 and 2, we will generate engineered nucleases to create DNA double strand breaks (DSBs) and indels at selected genomic sites for each target gene. The nucleases will be microinjected 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 co-target a pigmentation gene to serve as a mutagenesis selection marker (the selection marker was previously validated at CAT). Furthermore, we will preferentially raise batches of embryos in which mutations are produced at the one or two cell stage, (ie. detection of 2 or 4 mutant alleles per targeted loci by fragment analysis assay).We hypothesize that fish carrying double homozygous mutations that are deficient in spermiogenesis and steroid hormone production will develop as sterile males. Fertilization capacity of sperm will be assayed by in vitro fertilization of wild type eggs from 3 different females at the optimal sperm to egg ratio (100 eggs for 5.106 spermatozoa) and expressed as a percentage of surviving embryos to total eggs collected at 24hrs post fertilization. The mean values obtained from these studies will be compared across mutant genotypes using an unpaired t-test. Sperm density will be counted using a Neubauer hemocytometer slide and sperm motility will be measured in terms of percent motile spermatozoa in field of view.Objective 2. Evaluate two genes targeting male differentiation in conjunction with two other genes controlling oogenesis to produce a sterile all-female population (Years 1-3).In Objective 2, we will first generate mutations in six genes independently and cross selected F0 and F1 mutants to generate F2 double homozygous KO mutant lines.To assess sterility, a minimum of six double KO females for each genotype will be dissected at 4 and 6 months of age and their gonads photographed in situ before dissection. The mean total gonadosomatic index will be statistically compared across all genotypes relative to wildtype fish (unpaired T-test). Ovaries from 6-month old females showing no vitellogenic oocytes (stage II or III) will be fixed, dehydrated and embedded in paraffin for sectioning and stained with hematoxylin and eosin. The diameters of follicles with visible nuclei will be measured for staging under microscopic examination.Objective 3. Develop methodology to rescue male and female sterility, ensure propagation of the lines, and evaluate their performance (2-3 Years). In Objective 3, we will test if spermatogonial and oogonial stem cells harvested from the testis and ovaries of KO mutant all-male and all-female sterile tilapia, can be transplanted into the testes and ovaries of germ cell-less recipient adult tilapia and produce functional spermatozoa and oocytes carrying the donor genotype. We will select 2 different genotypes for each of the male and female transplantation recipients, using 5 recipient fish for each of the 4 genotypes tested. To evaluate the functionality of donor-derived gametes produced after transplantation, we will perform in vitro fertilization assays.Next, to measure the potential benefit of sterility, we will perform growth trials. Initially, embryos for each group (fertile control and sterile mutant) will be produced and reared separately. When fish within a group reach a mean weight of 60g, they will be PIT tagged and divided into six 1000L grow-out tanks, where they will be fed three times daily, to satiation. During the experiment, fish will be individually weighed, and the length of each fish will be measured at 4-week intervals. At the end of the experiment, when control groups reach market size (680g Sdv: 77g, 8 months), fish will be sacrificed and sexed based on the structure of the urogenital orifice. We will record the individual weights of dissected gonads and the carcass to calculate of gonadosomatic index (GSI) and carcass index (n=60 per group). Specific growth rate (G) will be calculated according to the formula of Houde & Scheckte.

Progress 09/01/21 to 08/31/22

Outputs
Target Audience:Our target audience includes aquaculture producers and suppliers, innovators, retail and foodservice buyers, government risk assessors and decision-makers, academia (research scientists, students, and other members of the scientific community), NGO and finance. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?This year, our results were presented to members of the scientific community and to aquaculture professionals through oral and poster presentations at six Aquaculture conferences (in North America and Europe). Our results were further presented to producers, seafood industry leaders, policy makers and NGOs through seminars (ACFFA's Aquaculture Research, Science and Technology Forum; Global Seafood Alliance), press articles (With tools like CRISPR, can genome editing deliver more resilience for aquaculture?) and video product (Overcoming the "sterility paradox") available on YouTube and accessible on our company's website. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? During the next three decades, humanity will need to double food production to feed a growing global population. Genetic engineering (GE) offers tremendous potential to increase farm productivity and finfish aquaculture represents an important solution for sustainable animal protein production. Growing infertile and monosex GE fish will reduce the potential negativeimpact of escapees on natural ecosystems and further improve productivity. In this program, we showed how to use germ cell transplantation methodology to propagate and mass produce infertile male only (objective 1&3) or female only GE tilapia populations (objective 2&3) at commercial scale. This past year, we examined the effect of sterility on growth (objective 3) by individually weighing sterile and fertile control sibling tilapia at monthly intervals from 3-10 months of age. We measured no changes in specific growth rate between 3 and 5 months from any GE-infertile and control fish of the same gender. However, after sexual maturation, we found that the specific growth rate of sterile tilapia increased significantly and gradually above control over time. Specifically, we measured a 13%**, 22%** and 50% increase in specific growth rate (SGR %/month) in sterile male versus fertile male between 7-8, 8-9, and 9-10 months of age respectively. We also found a 25%* and 34%* and 70%**increase in SGR in sterile versus fertile female between 7-8, 8-9, and 9-10 months of age. (**p<0.001, *p<0.05). Final total body weight was ~5% and ~10% larger in sterile versus fertile males and females respectively at 10 months of age. Overall, the growth performance of sterile tilapia was superior to their fertile sibling during the critical late growing phase. These results support the idea that energy channeled into the gonads of fertile fish detracts from somatic growth and indicate that blocking sexual maturation should confer significant benefits to commercial aquaculture producers. Finally, we believe this strategy can be transferred to any commercial finfish species and make aquaculture more profitable and environmentally friendly.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Oral Presentation: "Latest Developments in The Application of Genome Editing to Aquaculture Atlantic Canada Fish Farmer Association. X Lauth et al., ACFFAs Aquaculture Research, Science and Technology Forum. Oct. 2021
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Why do we need gene editing in Aquaculture Alan Tinch et al., Aquaculture UK Aviemore, Scotland May 5, 2022
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Oral Presentation: "SOLUTION FOR PROVIDING STERILE FARMED FISH X Lauth et al., Aquaculture 2022 conference, San Diego March 2022
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Oral Presentation: Genome Editing to Produce Monosex and Sterile Fish for Aquaculture, Buchanan et al, 6th Genomics in Aquaculture (GIA) Symposium Granada, Spain, 46 May 2022
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: "Report on the 6th Genomics in Aquaculture (GIA) Symposium Held in Granada, Spain, 46 May 2022." Aquaculture Journal 2(2): 72-163 (Ruiz-Rej�n, de la Herr�n et al. 2022)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Poster: "Genome Editing to Produce Monosex and Sterile Fish for Aquaculture" Advances in Genome Biology and Technology (AGBT) April 4-6 Coronado USA, 2022
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Poster "Three steps for making monosex, infertile fish" (EAS) Aquaculture Europe Sept 27-30, 2022, Rimini Italy
  • Type: Websites Status: Accepted Year Published: 2022 Citation: https://www.globalseafood.org/advocate/with-tools-like-crispr-can-genome-editing-deliver-more-resilience-for-aquaculture/


Progress 09/01/18 to 08/31/22

Outputs
Target Audience:Our target audience includes aquaculture producers and suppliers, innovators, retail and foodservice buyers, government risk assessors and decision-makers, academia (research scientists, students, and other members of the scientific community), NGO and finance. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Theproject has provided training and professional development opportunities for fourresearch associates and one student intern. The project further provided training on fish husbandry to four individuals with part-time positions. How have the results been disseminated to communities of interest?Our results were presented at over 12conferences or seminar,through oral and poster presentations addressed to the scientific community, students,aquaculture professionals, NGOs, government risk assessors and descion-makers, retail and foddservice buyers. Our results were further printed in abstract books, patent application,trade press and recorded in a shortvideo available online. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Food production will need to double over the next three decades to feed the world's growing population. In addition, climate change and economic crisis will continue to provide numerous challenges that innovative strategies like genetic engineering (GE) can help overcome. GE offers tremendous potential to increase fish farm productivity and finfish aquaculture represents an important solution for sustainable animal protein production. However, widespread adoption of GE fish is hampered by concerns over their accidental release to natural ecosystems. Growing infertile and monosex GE fish will reduce the potential negativeimpact of escapees on natural ecosystems and further improve productivity. In this program, we used GE and germ cell transplantation methodologies to generate and mass produce infertile male only (objective 1&3) or female only tilapia populations (objective 2&3) at commercial scale. This sterilization solution can be transferred to multiple fish species. Objective 1. To produce infertile males, we generated lines of tilapia carrying loss of function mutation in two categories of genes simultaneously. We inactivated genes essential for estrogen production (Cyp17 and Cyp19a1a) which caused genetic female to sex reverse into male. We further inactivated genes controlling spermiogenesis and selected mutant line with dramatic reduction in sperm production. Semen from these mutants contain low sperm density and high frequency of morphologically deformed spermatozoa. Female mutants for any one of those genes show no fertility defects. We then produced double homozygous mutants (sterile males) by first outcrossing to obtain compound heterozygotes followed by self-crossing to obtain the double homozygous mutant (e.g., Cyp19a1a -/-; Tjp1a-/-, Cyp17-/-; Gopc-/-). As expected, we found that all double-knockout tilapia (6% of the progeny population) developed as infertile males with defective sperm while full siblings with incomplete inactivation of the genes targeted developed as fertile males and females. Objective 2 Sterile females were successfully produced by blocking male differentiation and oogenesis. Specifically, we first created five lines of tilapia carrying loss of function mutation in two categories of genes simultaneously. The first mutation increases the synthesis of estrogen (Dmrt1-/- or Gsdf-/-) and causes genetic male to sex reverse into female. The second mutation blocks the production of functional oocytes (FSHR-/- or VTGAa-/- or VTGAb-/-) and results in female sterility. We then produced double homozygous mutants (sterile females) by outcrossing the FSHR, VTGAa and VTGAb heterozygotes (FSHR+/−, VGVTAa+/−, VTGAb+/−) with Gsdf heterozygous (Gsdf+/-) to obtain compound heterozygotes and followed by self-crossing to obtain the Gsdf-/-; FSHR-/-, Gsdf-/-; VTGAa-/- and Gsdf-/-; VTGAb−/−. As expected, we found that all double-knockout tilapia (6% of the progeny population) developed as sterile females while siblings with partial inactivation of the genes targeted developed as fertile males and females. Objective 3. To assess if infertile female only or male only populations can be produced at scale, we developed germ cell transplantation (GCT) procedures where recipient embryos can receive germ line stem cells extracted from the gonad of double KO fish. We harvested spermatogonial and/or oogonial stem cells from the gonad of any double KO lines created in objective 1 and 2 (Cyp17-/-, Cyp19a1a-/-, Dmrt1-/-, Gsdf-/-, FSHR-/-, VTGAa-/- and VTGAb-/-) and transplanted these cells into the peritoneal cavity of germ-cell-free recipient embryos (dnd1-/-; or Elavl2-/-). We consistently measured a colonization success rate exceeding 50% and found that recipients that received donor derived germ cell developed into male and female with a normal sex ratio and gametes production. Our results indicate that germline carrying null alleles of these genes retain their intrinsic capacity to proliferate, re-populate the gonads, and differentiate into functional sperm or eggs upon transplantation into a WT/permissive recipient. We confirmed that all recipient fish only produced donor derived mutant germ cells. Thus, our results demonstrate that somatic cells from the recipient fish provide a permissive environment for the mutant germ cell to develop into functional gamete of both sexes. We found that mating of the transplanted tilapia produced 100% sterile female offspring, or infertile male offspring as evidenced by their underdeveloped ovaries or spermatozoa depleted testis. Finally, we performed tank grow-out trials to compare the growth of full and/or half sibling tilapia with or without a functional gonad. Total Body Weight measurements were made at monthly intervals from 3-10 months of age. We measured no changes in specific growth rate between 3 and 5 months from any GE-infertile and control fish of the same gender. However, after sexual maturation, we found that the specific growth rate of sterile tilapia increased significantly and gradually above control over time. Specifically, we measured a 13%**, 22%** and 50% increase in specific growth rate (SGR %/month) in sterile male versus fertile male between 7-8, 8-9, and 9-10 months of age respectively. We also found a 25%* and 34%* and 70%**increase in SGR in sterile versus fertile female between 7-8, 8-9, and 9-10 months of age. (**p<0.001, *p<0.05). Final total body weight was ~5% and ~10% larger in sterile versus fertile males and females respectively at 10 months of age. Overall, the growth performance of sterile tilapia was superior to their fertile sibling during the critical late growing phase. These results support the idea that energy channeled into the gonads of fertile fish detracts from somatic growth and indicate that blocking sexual maturation should confer significant benefits to commercial aquaculture producers. Finally, we believe this strategy can be transferred to any commercial finfish species and make aquaculture more profitable and environmentally friendly.

Publications

  • Type: Websites Status: Accepted Year Published: 2022 Citation: https://www.globalseafood.org/advocate/with-tools-like-crispr-can-genome-editing-deliver-more-resilience-for-aquaculture/


Progress 09/01/20 to 08/31/21

Outputs
Target Audience:Our target audience includes aquaculture producers, innovators,government risk assessors and decision-makers, research scientists, students and other members of the scientific community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided training for a summer intern undergraduate from UC Davis and continued professional development opportunities for S. Herbert, BS and, who contributed to the molecular, morphological, and histological investigations. The project further provided training on fish husbandry to two individuals with part-time positions, including one BS student from UCSD. How have the results been disseminated to communities of interest?Our results were presented at in-person (Aquaculture America 2021, San Antonio) and at virtualconferences or seminar (International Conference of Animal Genetics 2021, Transgenic Animal Research Conference XIII, Aquaculture UK 2021) through oral and poster presentations. Our results were further printed in abstract books of the above-mentioned conferences and in our patent applications available online (Publication numbers: EP3833184A1 and US2021298276A1, publication dates: 2021-06-16 and 2021-09-30, " A METHOD OF GENERATING STERILE AND MONOSEX PROGENY"). What do you plan to do during the next reporting period to accomplish the goals?We requested 12 months, no cost extension for our Grant Award to quantify the potential benefit of sterility (i.e., growth performance, food conversion etc.) in tank grow-out trials (part of objective 3).

Impacts
What was accomplished under these goals? Advantageous traits have been successfully integrated in the most economically important aquaculture finfish species through genetic engineering (GE) technologies. Unfortunately, widespread adoption of these GE fish is hampered by concerns over their accidental release to natural ecosystems. Existing solutions to this problem remain technically difficult, costly, and insufficiently robust or scalable to be used in commercial settings. Here we provide methods for mass production of sex-specific sterilized finfish by disrupting their sexual differentiation and gametogenesis pathways. This approach is compatible with genetic breeding programs and will ensure biological containment of GE lines. Furthermore, production of monosex sterile fish will improve culture performance by reducing inbreeding during grow-out and ensuring size uniformity at harvest. Finally, monosex culture can capitalize on existing sexual dimorphic traits with economic value such as body size, color pattern and behavior. Objective 1: During this reporting period, we investigated the effect of estrogen deficiency on tilapia testicular activity. Zang et al. recently reported that loss of function mutations in Cyp19a1b cause male infertility by obstruction of the efferent duct. We created a cyp19a1bmutant line and acyp19a1a; cyp19a1bdouble-mutant line in tilapia. In contrast to earlier reporting, we found thatcyp19a1brelease sperm upon stripping. However, we found variable level of sterility expression among 10 months old Cyp19a1a-/-; Cyp19a1b-/- sibling males. We found that half of these males produced copious seminiferous secretion with spermatozoa concentration ranging from 1% to 0% (azoospermia) of control while the other halfproduced small volumes (5-10 folds reduction) of milt containing normal spermatozoa concentration (1x109 sperm/mL). Dissection of the gonadsreveals smaller testis suggesting that the reduced testicular secretion in double KO testis results from decrease seminiferous tubule luminal volume. The natural fertility rates of these estrogen deficient males will be interrogated. Objective 2: Sterile females were successfully produced by blocking male differentiation and oogenesis as previously reported. Objective 3: To assay if genetically sterile female populations can be produced, we first outcrossed theFSHR, VTGAa andVTGAbheterozygotes (FSHR+/−, VGVTAa+/−, VTGAb+/−) with Gsdf heterozygous (Gsdf+/-) to obtaincompound heterozygotes and followed by self-crossing to obtain the Gsdf−/−; FSHR−/−, Gsdf−/−; VTGAa−/− and Gsdf−/−; VTGAb−/−double homozygous mutants. As expected, we found that all double-knockout tilapia (6% of the progeny population) developed as sterile females while siblings with partial inactivation of the genes targeted developed as fertile males and females. Next, we transplanted oogonial stem cells from juvenile double KO-mutant donors into sterile recipient embryos. We found that transplanted recipients developed functional testis and ovaries. For each of the three double KO combinations tested, mating of the transplanted tilapia produced 100% sterile female offspring, as evidenced by their underdeveloped ovaries. Our results indicate that germline carrying null alleles of these genes retain their intrinsic capacity to proliferate, re-populate the ovaries, and differentiate into functional eggs upon transplantation into a WT/permissive recipient.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Oral Presentation W244-Genome editing to produce monosex and sterile fish for aquaculture, International Society for Animal Genetics (ISAG) 2021 Abstract Book p.45
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Poster Presentation P239-Genome editing to produce monosex and sterile fish for aquaculture, International Society for Animal Genetics (ISAG) 2021 Abstract Book p.88
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: A Solution for Providing Sterile Farmed Fish - (Video) Transgenic Animal Research Conference XIII - Day 2 - YouTube
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Genome editing to produce monosex and sterile fish for aquaculture, Aquaculture UK webinar in conjunction with the Center for Aquaculture Technologies
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Oral Presentation, MONOSEX AND STERILE FISH PRODUCED USING GENOME EDITING, Aquaculture America, San Antonio, TX, AA2021AbstractBook.pdf (windows.net) P.57


Progress 09/01/19 to 08/31/20

Outputs
Target Audience:Our target audience includes students, research scientists, and other members of the scientific community, as well as aquaculture producers, CAT industrial partners, 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 Dr. V. Williams, , M. Hoffman, MS, and S. Herbert, BS, who contributed to the molecular, gene editing, morphologcial and histological investigations (synthesis of engineered nucleases, microinjection of embryos, analysis of genotype and phenotype). 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/2020/033940, publication date: 13.02.2020, " A METHOD OF GENERATING STERILE AND MONOSEX PROGENY"). What do you plan to do during the next reporting period to accomplish the goals?We will generate Cyp19a1b-/-,Cyp17-/- mutants and analyze their sex and fertility (Objective 1). We will perform germ cell transplantation to generate male and female broodstock with normal estrogen production and gamete development. We will confirm that these broodstock can breed naturally and can produce sterile monosex population of progeny. We will assay the performance of these monosex progeny in tank grow-out trials (Objective 3).

Impacts
What was accomplished under these goals? Multiple species of fish have been genetically engineered (GE) to incorporate advantageous traits for aquaculture or to produce valuable pharmaceutical proteins. Unfortunately, widespread adoption of these GE fish is hampered by concerns over their accidental release to natural ecosystems. Existing solutions to this problem remain technically difficult, costly and insufficiently robust or scalable to be used in commercial settings. Here we provide methods for mass production of sex-specific sterilized finfish by disrupting their sexual differentiation and gametogenesis pathways. This approach is compatible with genetic breeding programs and will ensure biological containment of GE lines. Furthermore, production of monosex sterile fish will improve culture performance by reducing inbreeding during grow-out and ensuring size uniformity at harvest. Finally, monosex culture can capitalize on existing sexual dimorphic traits with economic value such as body size, color pattern and behavior. Objective 1. We created four lines of tilapia, each carrying loss of function mutations in two categories of genes. The first mutation blocks the synthesis of estrogen (Cyp17-/- or Cyp19a1a-/-) and the other causes sperm deformities (Tjp1a-/-, Csnk2a2-/-, Hiat1-/-, Smap2-/-). We found that double homozygous KO fish develop exclusively as male. However, we found that the sperm deformity phenotypes were insufficient to render these males fully sterile. We suspect that a mechanism known as genetic compensation is activated, where the transcription of a gene(s) related to the mutated gene is upregulated in mutants over time and functionally compensates for the mutated gene. To overcome this difficulty, we targeted a new gene Cyp19a1b whose inactivation was recently described to cause obstruction of the efferent duct in the male reproduction system. We selected a mutant allele of this gene that removes 80% of the protein sequence, bred this mutation into the Cyp17 mutant background and selected double hemizygous mutant. Objective 2. We have created five lines of tilapia carrying loss of function mutation in two categories of genes simultaneously. The first mutation increases the synthesis of estrogen (Dmrt1-/- or Gsdf-/-) and causes genetic male to sex reverse into female. The second mutation blocks the production of functional oocytes (FSHR-/- or VTGAa-/- or VTGAb-/-) and results in female sterility. A comprehensive phenotypic analysis of double KO mutants was made at month intervals over 6 months from 4 to 10 months of age. Data collected at the morphological, physiological, and histological levels confirmed that all double KOs are sterile females. Objective 3. We developed germ cell transplantation (GCT) procedures where hundreds of recipient embryos can receive donor derived germ cells in less than 1hr with an overall transplantation success rate of 50% to 90%. We harvested spermatogonial and/or oogonial stem cells from juvenile gonad of six single KO lines (Cyp17-/-, Cyp19a1a-/-, Dmrt1-/-, Gsdf-/-, FSHR-/-, VTGAa-/- and VTGAb-/-) and transplanted these cells into recipient embryo progeny from Elavl2-/+ or dnd1 +/- parents. Homozygous null mutant for Elavl2 and dnd1 are germ cell free fish whose gonad could nurse donor derived germ cells. We found that homozygous recipients (Elavl2-/- and dnd1-/-) that received Cyp17-/-, Cyp19a1a-/-, Dmrt1-/- or Gsdf-/- germ cell developed into fertile male and female with a normal sex ratio. We also found that recipient female that received VTGAa-/- or VTGAb-/- germ cell developed functional ovaries and produced healthy progeny. FSHR transplanted female have not yet sexually matured. We genotyped the progeny from transplanted fish and confirmed that all recipient fish only produced donor derived mutant germ cells. Altogether, our results demonstrate that somatic cells from the recipient fish provide a permissive environment for the mutant germ cell to develop into functional gamete of both sexes.

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 Lauth et al.,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 T. Umazume , M. Hoffman, V. Williams, J. Buchanan, X. 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: X. Lauth: "Gene editing to induce sterility in fish farming" special producer session for Aquaculture America 2020


Progress 09/01/18 to 08/31/19

Outputs
Target Audience:Professionals working in aquaculture breeding and genetics, policy makers and regulators in charge of measuring the risk benefit of GE technologies, scientists interested in the application of GE technologies to aquaculture species. Some aspect of this work will be presented at the Aquaculture America 2020 conference. Changes/Problems:The project is advancing with no significant problems or changes in planning. What opportunities for training and professional development has the project provided?The project has provided training and professional development for two individuals. Valerie Williams, phD and Melissa Hoffman, MS, have receive training on methods used to inactivate gene function in tilapia, which include the design and synthesis of engineered nucleases, embryo microinjection procedures, genotyping 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?A poster was presented at the Project Director's Meeting for Biotechnology Risk Assessment Grants (BRAG) Program (June 2019). Talks and poster presentations are scheduled for the coming year, including an oral presentation at the Aquaculture America 2020 conference. A patent application covering our strategy and proof of concept was filed and will be officially published in early February 2020. What do you plan to do during the next reporting period to accomplish the goals?We intend to generate a minimum of 10 double knockout (DKO) lines of tilapia, with one inactivated gene affecting sex differentiation and the other gamete function. These DKO fish should develop either exclusively as male or female and be reproductively sterile.

Impacts
What was accomplished under these goals? During this first year we generated 12 mutant lines of tilapia and analyzed their reproductive phenotypes and sex ratio . Objective 1. We have generated loss of function mutations in two genes deemed essential for female differentiation and confirmed that their inactivation cause genetic females to sex reverse into phenotypic males. Furthermore, we generated mutants in five genes modulating spermiogenesis. Semen from these mutants contain low sperm density and high frequency of morphologically deformed spermatozoa. Female mutants for any one of those genes show no fertility defects. We confirmed that at least 3 of those genes are expressed in the somatic gonad. Hemizygous mutants (F1 generations) for 3 of these genes have been generated. Objective 2. We have generated lines of tilapia carrying loss of function mutations in two genes governing male development. We found at least one inactivated gene causing male-to-female sex reversal. Hemizygous mutant lines have been produced for both genes. We further generated loss of function mutations in 3 genes deemed essential for oogenesis and found that their inactivation cause ovarian failure or result in females producing non-viable embryos. Males carrying the same gene mutations were fertile. A total of 5 single and two double hemizygous mutant lines have been generated as part of this objective.

Publications