Progress 07/01/20 to 06/30/23
Outputs
Target Audience:This project is mostly technical and presentations were given at scientific meetings of people working in this field. Additionally, during the past year the PD gave thirty public presentations to stakeholders and audiences throughout the world. The lectures were as follows: 1. "The need for harmonized, risk-based regulation of gene editing in livestock", 12th World Congress of Genetics Applied to Livestock Production, Rotterdam, The Netherlands 7/5/2022 2. "History and author analysis of the World Congresses on Genetics Applied to Livestock Production" 12th World Congress of Genetics Applied to Livestock Production, Rotterdam, The Netherlands 7/5/2022 3. "Gene editing in livestock: Science and policy" Third Symposium on Synthetic Biology in the series "Harnessing Transformational Technologies" Virtual, Lawrence Livermore National Lab 7/12/2022 4. "Synergistic power of genomic selection, assisted reproductive technologies, and gene editing to drive genetic improvement of cattle" QAAFI science seminar, virtual, https://youtu.be/KODZxdtjwKc 8/2/2022 5. "Genome Editing Opportunities in Agriculture: Perspectives on Livestock Breeding", Genome Web, virtual, 8/6/2022 6. "Genome Editing", Simmental Fall Focus, Roanoke, VA 8/27/2022 7. "Current State of Genome Editing and What is Means for Producers" Applied Reproductive Strategies Applied to Beef Cattle (ARSBC), San Antonio, TX 8/31/2022 8. "Communicating with the general public about GMOs and genome edited organisms", 4th International Workshop on Regulatory Approaches for Agricultural Applications of Animal Biotechnologies, São Paulo, Brazil, 9/13/2022 9. "The History and Impact of Misinformation in the Agricultural Sciences", Missouri Farm Bureau's Advanced Leadership Academy, Virtual lecture, 9/19/2022 10. "Alternative meats and alternative facts", American Association of Bovine Practitioners, Long Beach, CA 9/24/2022 11. "Synergistic power of genomic selection, assisted reproductive technologies, and gene editing to drive genetic improvement of cattle", 50 min virtual seminar for BMS 792 Seminar class, Animal Reproduction and Biotechnology Laboratory, Colorado State University 9/26/2022 12. "The Importance of Genetic Improvements to the Future Sustainability of Animal Agriculture", 48th National Academy of Sciences, Republic of Korea International Symposium, Seoul, South Korea, 10/14/2022 13. "Synergistic power of genomic selection, assisted reproductive technologies, and gene editing to drive genetic improvement of cattle", Seoul National University (SNU), Republic of Korea, 10/17/2022 14. "The Importance of Genetic Improvements to the Future Sustainability of Animal Agriculture", New Paradigm of Animal Agriculture by Utilization Biotechnology and Bioresource in Developing Countries, Chungbuk National University, Republic of Korea, 10/18/2022 15. Animal Genomics Enables Selection and Dissemination of Improved Livestock", Keynote at Australasian Genomic Technologies Association (AGTA) 2022 Conference, Twin Waters QLD, Australia 10/31/2022 16. "Science Friction: Can effective communication save genome editing from the fate of GMOs?", 2022 D.W. Brooks Lecture, University of Georgia Athens, GA. 11/8/2022 17. "Genome Editing Opportunities in Livestock", Plant and Animal Genome Conference, San Diego, CA 1/14/2023 18. "Animal Agriculture and Alternative Meats: Learning from Past Science Communication Failures" Virtual, 2023 Pacific Northwest Animal Nutrition Conference 1/17/2023 19. "Global overview of animal biotechnology applications in the livestock industry", Virtual, Biotechnology Applications and Impact on the Philippine Livestock Industry, The Philippines 1/30/2023 20. "Global overview of animal biotechnology applications in the livestock industry" UC Davis Department of Animal Science Seminar, 2/6/2023 21. "Global Overview of Animal Biotechnology for Climate Change Resiliency, Food, & Agriculture", Virtual, Building knowledge and regulatory capacity in animal (livestock and aquaculture) biotech (GE and GnEd) in response to climate change. Asia-Pacific Economic Cooperation High Level Policy Dialogue on Agricultural Biotechnology (APEC HLPDAB) 2/28/2023 22. "Genome Editing Opportunities in Livestock" Beef Club, UC Davis, Davis, CA 2/28/2023 23. "Outcomes from embryonic editing of livestock genomes", AGBT Ag, San Antonio, TX 4/27/2023 24. "Animal genomics and Biotechnology Applications in the Livestock Industry", National Academy of Sciences Workshop on Climate Impacts and Sustainable Agriculture, Irvine, CA 4/11/2023 25. "Effective Extension within a Research Program", Getting Research Into Practice Workshop, Elora, Ontario, Canada 4/19/2023 26. "Alternative Meats and Alternative Metrics" International Society for Biosafety Research (ISBR), St. Louis, MO 5/1/2023 27. "What is a Sustainable Biotechnology?", International Society for Biosafety Research (ISBR), St. Louis, MO 5/4/2023 28. "Use of gene editing technologies in livestock production systems", Genomics-assisted breeding for boosting crop and livestock improvement ", Barcelona, Spain 5/11/2023 29. "Is Agricultural Innovation the Solution or the Problem?", Dairy Symposium, Madison, WI 5/18/2023 30. "Emerging reproductive technologies in ruminant production", International Ruminant Reproduction Symposium (IRRS), Galway, Ireland 6/1/2023 Changes/Problems:The COVID-19 pandemic and supply chain issues slowed progress on this project, we therefore obtained a no-cost extension to complete the work outlined in the grant. As a result of delays during the stay-at- home orders, expenditures on personnel were higher than originally budgeted whereas expenditures on supplies and travel were less than originally budgeted due to period when wet laboratory work was not allowed. What opportunities for training and professional development has the project provided?A Master's student was involved in the original research for this project and published the results of his thesis in the 2023 peer-reviewed paper and has now moved onto a job in the biotechnology industry. Additionally four undergraduate students were employed or served as interns on this project. Two of these undergraduate graduating students subsequently obtained jobs as research technicians in biotechnology companies. Moreover, two newer Master's students in the laboratory have been trained on using the electroporation methods optimized in this study to perform genome editing in sheep with very high success rates, and the resulting edited lambs will be the basis of their Master's research projects. How have the results been disseminated to communities of interest?As an extension specialist a considerable amount of my time is spent communicating the results of the research that my laboratory performs, and more generally the concepts of gene editing in livestock to the general public. Despite COVID, over the course of the three years of this grant I have given over 100 presentations to audiences throughout the globe. Needless to say the pandemic decreased the opportunities to give in person presentations, but after lock-down I worked with a disparate variety of groups around the globe to pivot my educational materials and presentations to virtual content including UC Berkeley University Extension, Zoetis Dairyman's Roundtable, Montana Nutrition Conference, Beef Improvement Federation, International Food Information Council Webinar Series, K-12 science educators teacher training with American Farm Bureau Foundation for Agriculture, International Food Technology Institute Virtual Conference, Genome Writer's Guild, International Consortium on Applied Bioeconomy Research (ICABR), Arizona State University Animal Science Department, University of Kentucky, Davis Sr High women in Science, Technology, Engineering, and Mathematics (WiSTEM), Food Drug and Law Institute (FDLI), Syntego World CRISPR Day symposium, Iowa State seminar, Grains and Health Symposia, Calgary, Transgenic Technology, CAST Annual meeting, ASAS-Southern Section Genetics and Genomics Webinar Series, Virtual Workshop in Genome Editing Technologies in Kenya, Wagyu Virtual International Conference, South Africa, Centre for Genetic Improvement of Livestock (CGIL) Seminar, Department of Animal Biosciences, University of Guelph, Canada, National Center for genome editing in agriculture, Israel, and the British Cattle Breeders Club. The ability to present remotely opened up the opportunity to present to a larger and more diverse audience than typically attends in person meetings, and I spoke to audiences in countries that typically would not be able to support the cost of an in-person presentation (e.g. Kenya). I spoke at the 2021 USDA Agricultural Outlook Forum, American Angus Association, Global Roundtable for Sustainable Beef, Warnick Lecture, University of Florida, Golden State Dairy Management Virtual webinar, American Association of Bovine Practitioners, 4th International Workshop on Regulatory Approaches for Agricultural Applications of Animal Biotechnologies, National Academy of Sciences Workshop on Climate Impacts and Sustainable Agriculture, and was the first ever woman to be invited to present the Hammond Lectureship to the British Society of Animal Science. I organized and spoke at the UC ANR Livestock and Dairy Program Team meeting on Animal Agriculture and the Environment, gave the opening keynote at the Animal Agriculture Alliance; spoke at Brac University, Bangladesh; Dalhousie University, NS, Canada; Faculty of Animal Science, Ithaca, NY; Society for In Vitro Biology's Virtual Annual Meeting, SIVB 2021: In Vitro OnLine; the 2021 ASAS-CSAS-SSASAS Annual Meeting and Trade Show, The International Service for the Acquisition of Agri-biotech Applications Impact of Gene Technology in Animal Agriculture and Food Production; Royan Virtual Congress 2021, Iran; Agricultural Science Association (ASA) Ireland; IFIC Agricultural Technology and Food Production Salon; Australasian Genomic Technologies Association (AGTA) 2022 Conference, and the 2023 International Ruminant Reproduction Symposium (IRRS) in Galway, Ireland. I received the and the 2021 Beef Improvement Association (BIF) Pioneer Award awarded to those who have made lasting contributions to the genetic improvement of beef cattle and who have had a major role in acceptance of performance reporting and documentation as the primary means to make genetic change in beef cattle. As it relates to this project we prepared a detailed proceedings paper entitled "Current State of Genome Editing and what it means for Beef Producers" summarizing this project for the Proceedings of the Applied Reproductive Strategies in Beef Cattle August 30-31, 2022; San Antonio, TX https://beefrepro.org/wp-content/uploads/2022/08/2022-ARSCB-VanEenennaam-Mueller-Proceedings.pdf I continue to disseminate information through eBEEF, the eXtension beef genetics/genomics community of practice I lead to meet beef producers and Extension educators' needs in beef genetics programming. Reductions in beef genetics Extension specialists and outreach funding in the US led a group of beef genetics Extension faculty in Kentucky, Kansas, Missouri, Tennessee and California to coordinate their efforts and develop a national Extension program. This effort, collectively called eBEEF, has utilized four platforms to provide these outreach efforts: Beef Improvement Federation, National Beef Cattle Evaluation Consortium, eBEEF.org website (https://ebeef.ucdavis.edu), and the National Cattlemen's Beef Association education program. Materials provided through eBEEF include the publication of a 2021 revised version of the Beef Cattle Beef Sire Selection Manual which included my updated chapter on "DNA-based Technologies", and that was produced and distributed to cattle producers throughout the nation, major contributions to the Beef Improvement Federation Guidelines for Uniform Beef Improvement Programs, 32 factsheets, 165 videos, over 18 train-the-trainer webinar series, 5 direct producer education webinars, and numerous in person educational programs. This collaboration has proved to be an effective model to provide beef genetics outreach programming to a national audience. The 2021 BIF Ambassador Award was awarded to eBEEF for efforts in extending the news of BIF and its principles to a larger audience. I personally maintain and manage the eBEEF.org website and all aspects of my lab website (http://animalbiotech.ucdavis.edu) which averages approximately 2,000 visits monthly; my UC Davis BioBeef BLOG site (http://biobeef.faculty.ucdavis.edu), and my Twitter account (@BioBeef) which has more than 10,700 followers. I work extensively with print, radio, and television media and was featured on a variety of local and national programs to amplify the results of my research and reach the wider community. In past three years I participated in 284 media outlet/program interviews including a feature on the PBS NOVA "Gene Editing Reality Check" https://www.youtube.com/watch?v=E8vi_PdGrKg; and being cited in multiple articles (e.g. Forbes) on proposed regulatory approaches for genome edited livestock. These efforts helped to raise awareness about the need to invoke science-based regulation around genome editing in livestock. In December 2020, the USDA announced an advanced notice of proposed rulemaking that will transition portions of FDA's pre-existing animal biotechnology regulatory oversight to USDA, and in 2022 FDA gave regulatory discretion to two genome edited cattle. I have written and spoken extensively on this topic, including a recent editorial in Gen Biotechnology entitled "What is a Drug? Enabling the Bioeconomy Through Updated Food & Drug Administration Policy." What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We were ultimately able to optimize electroporation conditions for bovine, porcine and ovine zygotes for the efficient (>95% knockout) generation of genome-edited knockout blastocysts in livestock species. We had poor success rates with caprine oocytes surviving. Moreover, the electroporation methods developed in this project to genome edit sheep blastocysts with a very high efficiency have been employed in two ongoing projects targeting distinct genes in sheep. One of these applications is hypothesized to produce disease resistant sheep for agricultural applications, and the other to develop an ovine preclinical medical model for Pulmonary Arterial Hypertension (PAH), a rare vascular disorder occurring in 1-2 people per million individuals. Although these projects were beyond the original scope of the grant, in both cases using the conditions developed in this project, we achieved ~ 90% knockout efficiency and high rates of pregnancy viability following embryo transfer of the edited blastocysts to synchronized recipient ewes. AIM 1. Develop a platform for efficient targeted gene knockout using electroporation of the CRISPR/Cas9 system zygotes across four important food animal species. This project was funded right at the beginning of the Covid19 pandemic making it impossible to source ovaries from abattoirs for over a year. During this time the graduate student working on this project published a literature review entitled "Electroporation-Mediated Genome Editing of Livestock Zygotes". We were ultimately able to optimize electroporation conditions for bovine, porcine and ovine zygotes for the efficient (>95% knockout) generation of genome-edited knockout blastocysts. We had poor success rates with caprine oocytes surviving. In one ongoing project, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated system protein 9 (Cas9) system and a single guide RNA (sgRNA) approach was used to knockout a targeted gene in sheep embryos. Three sgRNAs targeting the gene were selected using software called Chop-Chop and electroporated as Cas-gRNA ribonucleoproteins into sheep zygotes 6-hours post fertilization using the conditions developed in this project. Blastocysts were genotyped by PCR amplification followed by Sanger Sequencing. Guide 3 was chosen due to the highest knockout efficiency (~90%), and the lack of homology to potential off-target sites in the reference ovine genome. Embryos were produced using abattoir derived oocytes and a total 55 edited blastocysts were transferred to 11 surrogate ewes using established embryo transfer techniques, resulting in 6 pregnancies and 11 embryos. Twelve blastocysts not transferred showed a knockout efficiency of 83%. Currently, five ewes carrying 8 fetuses are due to lamb in August, 2023. Following birth, genotyping and weaning; knockout and unedited control lambs will be subject to a disease challenge to determine if the knockout animals are more resistant to the disease. In another ongoing project, an experiment was performed to develop an ovine preclinical genetic model for Pulmonary Arterial Hypertension (PAH), a rare vascular disorder occurring in 1-2 people per million individuals. Monoallelic mutations in the bone morphogenetic protein receptor type 2 (BMPR2) gene are the most common genetic risk factor, with about 20% of carriers affected. BMPR2 is a serine/threonine kinase receptor in the transforming growth factor beta superfamily. Although autosomal dominant, BMPR2 has variable penetrance, pathologically expressing in ~42% of females and 14% of males. Despite well-known associations with PAH, the role of BMPR2 in the disorder's progression remains poorly understood due in part to the lack of appropriate preclinical genetic models. The homozygous BMPR2 (-/-) knockout genotype is embryonic lethal, which is an obstacle to creating a suitable genetic biomedical model for PAH. To overcome this barrier, a single guide RNA (sgRNA) CRISPR/Cas9 approach was used to edit sheep zygotes with the intent of creating a monoallelic knockout or missense mutation of a critical amino acid in exon 3 of the ovine BMPR2 gene. A single-stranded oligodeoxynucleotide (ssODN) that disrupted the PAM site was designed to serve as a synonymous repair template. Using this approach heterozygote lambs could be generated in two ways, either through high editing efficiency coupled with ssODN homology-directed repair (HDR) of one of the edited alleles, or intermediate editing efficiency with low to no presence of HDR in the genetic sequence. Ovine zygotes were electroporated six hours post-fertilization using sgRNA (100 ng/µL)/Cas9 (200 ng/µL) ribonucleoprotein complexes and three ssODN concentrations (100, 300, and 600 ng/ul). At seven days, zygotes that had advanced to blastocysts were lysed, PCR-amplified, and Sanger sequenced. Sequences were analyzed using TIDER, a computer model that quantifies the frequency of targeted small nucleotide changes introduced by CRISPR in combination with HDR using a donor template. A Kruskal-Wallis test was used to compare the efficacy of HDR at 100, 300, and 600 ng/ul of ssODN template; and 600 ng/ul resulted in highest rate of HDR (p < 0.0001). These conditions resulted in an editing efficiency of 44.7% high ( >66%), 42.6% intermediate (66-33%), and 8.5% low (<33%) knockout rate, and 4.2% wild-type; and an HDR knockin rate of 27.7% high (>20%), 21.3% intermediate (20-5%), and 51% low to no (<5%). Collectively, these data indicated that approximately half of the embryos subjected to these conditions would have the desired heterozygous BMPR2 (+/-) genotype, and so embryos were produced for embryo transfer. Five or six edited blastocysts were transferred to each of 8 recipient ewes resulting in 7 pregnancies, one of which subsequently failed. Eight fetuses (two sets of twins and four singletons) were confirmed viable via ultrasound at 9.5 weeks, past the embryonic lethality period, and the resulting lambs are expected to be born late in July 2023. DNA will be collected and analyzed for BMPR2 genotype. We predict that half of the lambs will be heterozygous BMPR2 (+/-). Semen from heterozygous males will be frozen to produce BMPR2 control (+/+) and heterozygous (+/-) lambs to serve as an ovine preclinical genetic model for heritable PAH. AIM 2. Develop a platform for efficient targeted gene knock-in using electroporation of the CRISPR/Cas9 system zygotes across four important food animal species Transfer of large DNA plasmids into the zygote is hindered by the zona pellucida (ZP). A potentially scalable approach to deliver HDR templates of up to 4.9 kb into zygotes is to use recombinant adeno-associated virus (rAAV) vectors. In this study, electroporation conditions were optimized to efficiently deliver CRISPR-associated protein 9 (Cas9):single guide RNA (sgRNA) ribonucleoprotein (RNP) complexes to bovine and ovine zygotes, while retaining embryo viability. rAAV vectors were then used to transduce a 3.9-kb HDR template in combination with electroporation of Cas9:sgRNA RNP editing reagents 6 h post insemination to generate bovine blastocysts with a targeted 2.7 kb knock-in (KI) at theH11safe harbor locus. With this approach, there was no need to remove or weaken the ZP of the blastocysts that developed, and a KI rate of up to ∼38% was observed. However, to circumvent transduction of the cumulus cells, the oocytes were denuded before transduction that decreased the blastocyst development rate. In addition, genetic mosaicism was observed in the blastocysts. These results highlight that further optimization of editing approaches will be required to achieve nonmosaic embryos with a targeted KI at scale for livestock applications. Given the low success rate in bovine embryos this platform was not tried in other species.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Van Eenennaam, A. L. 2023. What is a Drug? Enabling the Bioeconomy Through Updated Food & Drug Administration Policy. GEN Biotechnology. 2(1) https://doi.org/10.1089/genbio.2023.0003
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Lin, J.C., T.F. Bishop, J.R. Owen, S.L. Hennig, D.S. Fitzpatrick, M.L. Mueller, D. Guan, M.L. Angove, A. Hua, E.G. Balogh, R. J. Swift, P.J. Ross, and A.L. Van Eenennaam Generation of 2.7 kb Knock-in Bovine Blastocysts with Electroporation and Adeno-Associated Virus Delivery of CRISPR Reagents. April 27-29, 2023 AGBT Ag, San Antonio, TX
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Progress 07/01/21 to 06/30/22
Outputs
Target Audience:This project is mostly technical and presentations were given at scientific meetings of people working in this field. Additionally, the PD gave twenty-seven public lectures regarding genome editing in livestock were presented to stakeholders and audiences throughout the world. The lectures were as follows: 1. "Gene editing in livestock: Science and policy" Academy of Veterinary Consultants (AVC), Amarillo, TX 8/6/2021 2. "Gene Editing in Livestock: What is Gene Editing?" Idaho State University Virtual Event, 8/21/2021 3. "Effective Science Communication to Raise Awareness on Animal Biotechnology", The International Service for the Acquisition of Agri-biotech Applications (ISAAA) Impact of Gene Technology in Animal Agriculture and Food Production Virtual Workshop, 8/31/2021 4. "Genome Editing Opportunities in Livestock", Royan Virtual Congress 2021, Iran (virtual) 9/3/2021 5. "The Interdependence of Sustainability, Innovation, & Science Communication Around Animal Agriculture" Agricultural Science Association (ASA) Ireland, (virtual) 9/10/2021 6. "When Biotech Meets Animal Agriculture" IFIC Agricultural Technology and Food Production Salon, (virtual) 10/7/2021 7. "The Importance of Genetic Improvement to the Sustainability of Animal Agriculture", Roy Wallace symposium, Select Sires, Ohio 10/19/2022 8. "The Interdependence of Sustainability, Innovation, and Science Communication Around Animal Agriculture" Fort Hays State University, Hays, KS 11/2/2021 9. "Public Acceptance of Animal Genomics and Biotechnology", Visions III: Star Gazing into the Galaxy of Animal Genetics and Genomics. Iowa State University, Ames, IA 11/4/2021 10. "Genome Editing Opportunities in Livestock", Cultivating Trust Conference 2021, Farm and Food Care, Saskatchewan, Canada 11/18/2021 11. "Gene Editing Today and in the Future", Purina virtual on-going, continuing education training for the field sales staff, 11/30/2021 12. "Recent Changes in Genetic Technologies Acceptance & Regulation", The Poultry Breeders Roundtable (PBR) and National Swine Improvement Federation (NSIF) joint meeting, St. Louis, MO 11/30/2021 13. "Producing More from Less: the Role of the Efficient Animal", Net Zero Carbon: Meeting UK Livestock Targets, BSAS 12/3/2021 14. "Gene editing: What is it, and will US farmers and ranchers be able to use it?" South Carolina Farm Bureau Annual Meeting, Myrtle Beach, SC 12/3/2021 15. "Agriculture, Environmental Sustainability and Climate Change", California Feed and Grain Annual Meeting, Seaside, CA 1/13/2022 16. "Current & Future Genetic Research & Expectations", A Cow-Calf Symposium for Commercial Western Ranchers, Organized by the AZ Cattle Industry R & Ed Foundation (ACIF), Phoenix, AZ 2/18/2022 17. "Update on how genomics is being incorporated into breed association genetic evaluations", UCCE Siskiyou County Pie Night, Montague, CA 2/24/2022 18. "Genomics in the Dairy Industry: Progress and Prospects" UCCE Dairy Conference, Modesto, California 3/23/2022 19. "Science communication: Nagating Controversial Issues" Women in Stem Reverse Science Fair, WiSTEM club, Boston High School, Boston, MAVirtual 4/9/2022 20. "The importance of genetic improvement to the sustainability of dairy production systems" Genetic Strategies for Sustainability, California Dairy Sustainability Virtual Summit, 4/14/2022 21. "The future of biotechnology in impacting food insecurity", Iowa State, Des Moines, Iowa 4/20/2022 22. "Using genome editing for livestock health", Mahr Lecture on One-Health in the College of Veterinary Medicine Des Mones, Iowa 4/21/2022 23. "Synergistic power of genomic selection, assisted reproductive technologies, and gene editing to drive genetic improvement of cattle" World Jersey Cattle Bureau - Global Online Conference 4/28/2022 24. "Using cutting edge technology in dairy cattle breeding" ADSA Discover Conference, Itasca, IL 6/2/2022 25. "Emerging Technologies: Regulatory Oversight of Intentional Genomic Alterations in Animals", Food Drug and Law Institute (FDLI) Online Conference 6/14/2022 26. "Synergistic power of genomic selection, assisted reproductive technologies, and gene editing to drive genetic improvement of cattle", Production Animal Consultation Veterinary meeting, Hays, KS 6/15/2022 27. "Emerging reproduction technologies" ADSA Annual Meeting, Kansas City, KS 6/21/2022 Changes/Problems:The COVID-19 pandemic and supply chain issues slowed progress on this project, we therefore obtained a no-cost extension to complete the work outlined in the grant. What opportunities for training and professional development has the project provided?A Master's student was involved in the research for this project and published his Master's thesis this year, and has now moved onto a job in industry. Additionally four undergraduate students were employed or interns on this project. Two of these undergraduate graduating students subsequently obtained jobs as research technicians in companies. How have the results been disseminated to communities of interest?Presentations were given at the Transgenic Animal Conference, Plant and Animal Genome Conference - both virutally, and in person at the Large Animal Genetic Engineering Summit, . Park City, Utah. June 5-8, 2022. What do you plan to do during the next reporting period to accomplish the goals?In the next year we plan to finish optimizing the conditions for the electroporation of goat zygotes, and further refine methods to achieve targeted gene knock-ins using electroporation of embryos.
Impacts
What was accomplished under these goals?
AIM 1. Develop a platform for efficient targeted gene knockout using electroporation of the CRISPR/Cas9 system in MII oocytes and zygotes across four important food animal species (80% complete). We were able to optimize electroporation conditions for bovine, porcine and ovine zygotes for the efficient (>95% knockout) generation of genome-edited H11 knockout blastocysts. We had poor success rates with caprine oocytes surviving. . Based on mouse embryo electroporation literature, parameters including poring pulse voltage (5-40V), number (2-9), length (1-50msec), decay (0-40%), and polarity (bipolar or unipolar) and the Super Electroporator NEPA 21 (NEPA GENE, Chiba, Japan) were used to electroporate batches of 30-100 activated in vitro matured oocytes or fertilized sheep and cattle zygotes with Cas9 (100ng/µL)-single guide RNA (200ng/µL) ribonucleoprotein (RNP) complex targeting the H11 locus, a "safe harbor" site. Two bipolar 3.5msec pulses, 50msec interval, and 0% decay at 30V resulted in a 14% blastocyst development rate (N=537), and 97% mutation rate (N=37); while these same parameters at 40V resulted in a 18% blastocyst development rate (N=364), and 89% mutation rate (N=45) for sheep embryos. Three bipolar 3msec pulses, 50msec interval, and 0% decay at 20V resulted in a 31% blastocyst development rate (N=270), and 100% mutation rate (N=23) for cattle embryos. These conditions had higher mutation rates than others tested (P<0.05), but concomitantly decreased blastocyst development rates (P<0.05) as compared to controls (35.3% for 30V, N=398; 40.2% for 40V, N=308; 43.3% for 20V, N=210). These conditions achieve both high mutation rates and an acceptable rate of blastocyst development. AIM 2. Develop a platform for efficient targeted gene knock-in using electroporation of the CRISPR/Cas9 system in MII oocytes and zygotes across four important food animal species (50% complete). Transfer of large DNA plasmids into the zygote is hindered by the zona pellucida (ZP). A promising scalable approach to deliver larger donor templates for homology-directed repair of up to 4.7 kb along with genome editing reagents into zygotes, without using cytoplasmic injection, is to use recombinant adeno-associated viruses (rAAVs) in combination with electroporation. The objective of this project was to generate bovine blastocysts with a 2.7 kb knock-in at the H11 locus using electroporation and rAAV transduction. To characterize the native capacity of rAAV vectors to penetrate the ZP and deliver the donor DNA template into bovine zygotes, we transduced bovine zygotes with a panel of six natural rAAV serotypes (1, 2, 5, 6, 8, and 9) packaged with a CMV-eGFP reporter (Charles River, Rockville, MD), and screened blastocysts for GFP expression using a fluorescent microscope. Serotype rAAV6 was identified to efficiently transduce the ZP of bovine zygotes. We constructed a 3.9 kb donor cassette including 600 bp H11 homology arms with gRNA target sites at each end, the CAG promoter driving superfolder GFP with a nuclear localization signal, and rAAV inverted terminal repeat arms. This cassette was packaged into serotype rAAV6 and incubated with matured, denuded bovine oocytes at various concentrations for 6 hours during fertilization. Previously optimized electroporation parameters (20V, 3 bipolar pulses, 3.5msec pulse length, 50msec intervals, 0% decay rate with the Super Electroporator NEPA 21 (NEPA GENE, Chiba, Japan)), were subsequently used to transfect Cas9 (100ng/µL)-gRNA (200ng/µL) RNPs targeting the H11 locus into bovine zygotes. Zygotes were cultured for seven days, and blastocysts expressing GFP were selected, lysed, and underwent 2 rounds of PCR using 2 sets of junction primers flanking either the left or right homology arms. Sanger sequencing of PCR products confirmed the targeted knock-ins of the donor cassette in the green blastocysts. Knock-in and blastocysts rates were 35.7% (n=14), 6.7% (n=209) for rAAV6 at a concentration of at 8x1010 viral genome copies (vgc); 26.3% (n=19), 8.6% (n=222) for rAAV6 at a concentration of 9x1010 vgc; and 23.1% (n=13), 3.7% (n=351) for rAAV6 at a concentration of 1011 vgc, respectively.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Fitzpatrick, D.S., Ledesma, A.V., Lin, J.C., Datar, S.A., and A.L. Van Eenennaam. 2022. Targeted Inactivation of BMPR2 in Sheep Zygotes Using CRISPR/Cas9 Genome Editing. Large Animal Genetic Engineering Summit, . Park City, Utah. June 5-8, 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Lin, J. C., Bishop, T.F., Owen, J. R., Hennig, S. L., Hua, A., Swift, R.J., and A. L. Van Eenennaam. 2022 Using Electroporation and Adeno-Associated Virus Delivery of Editing Reagents to Generate a 2.7 kb Knock-in in Bovine Zygotes. Large Animal Genetic Engineering Summit, . Park City, Utah. June 5-8, 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Lin, J. C., Owen, J. R., Hennig, S. L., and A. L. Van Eenennaam. 2022. Optimization of Electroporation Parameters for the Introduction of Genome Editing Reagents into Early Sheep and Cattle Embryos. Plant and Animal Genome XXIX Conference Abstract. https://pag.confex.com/pag/xxix/meetingapp.cgi/Paper/45067
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2022
Citation:
Lin, J. C. Scalable Production of Genome-Edited Livestock Embryos. 2022. Master's Thesis. University of California, Davis. UC Davis Electronic Theses and Dissertations. 98 pages.
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Progress 07/01/20 to 06/30/21
Outputs
Target Audience:Twenty-two public lectures regarding genome editing in livestock were presented - mostly virtually - to stakeholders and audiences throughout the world. The target audiences included livestock producers both in the US and abroad, researchers working in genome editing, and the general public. Additionally the press picked up on some of these presentations and further amplified the information to their readership. This included a 8 minute feature on PBS' NOVA program on "CRISPR Gene-Editing Reality Check" which premiered September 9, 2020. Changes/Problems:The COVID-19 pandemic slowed progress on this project, especially as we were unable to access abattoirs to retrieve ovaries for oocyte collection for a number of months. Additionally we were unable to perform in person research in the laboratory for most of 2020. It is likely we will need to have a no-cost extension of a year to complete the work outlined in the grant. What opportunities for training and professional development has the project provided?A Master's student was involved in the research for this project and wrote a literature review during the COVID lockdown. Additionally two undergraduate students were employed to collect ovaries for this project once we were able to access the abbatoirs. One of these graduating students subsequently obtained an internship position at a commercial bovine embryo laboratory. How have the results been disseminated to communities of interest?A review paper on "Electroporation-mediated genome editing of mammalian zygotes" was published in Frontiers in Genetics. Twenty-two public lectures regarding genome editing in livestock were presented - mostly virtually - to stakeholders and audiences throughout the world. An in person keynote presentation on Gene Editing was given to the Beef Cattle Improvement Federation Annual Research Symposium and Convention in Des Moines, Iowa in June 2021, and a proceedings paper was prepared that accompanied the presentation. The lectures were as follows: 1. "Genome Engineering for Agricultural Applications" Genome Writer's Guild, 7/23/2020 https://youtu.be/OP6nUoZbv3U 2. "Potential of CRISPR in Livestock" International Consortium on Applied Bioeconomy Research (ICABR) Annual Program Online 7/24/2020 3. "Gene editing in livestock: promise, prospects and policy" Arizona State University Animal Science Departmental Seminar Online 9/21/2020 4. "Advanced Genetic Technologies" Reaching Out While Locked In! Beef Management Webinar Series, University of Kentucky Online 9/22/2020 5. "Genome Editing Approaches for Livestock --Advancing Animal Health and Welfare" Davis Sr High women in Science, Technology, Engineering, and Mathematics (WiSTEM) Online 9/29/2020 7. "Emerging Technologies: Regulatory Oversight of Intentional Genomic Alterations in Animals", Food Drug and Law Institute (FDLI) Online Conference 10/7/2020 8. "Genome Editing Applications in Animals" CRISPR in Agriculture Research, Syntego World CRISPR Day symposium 10/20/2020 9. "Gene editing in livestock: promise, prospects and policy" Iowa State virtual seminar 10/21/2020 10. "Agricultural animal transgenesis for food applications" Transgenic Technology, Israel, 10/27/2020 11. "Using genome editing for livestock health", ASAS-Southern Section Genetics and Genomics Webinar Series 11/4/2020 12. "Genome editing applications in animals" Virtual Workshop in Genome Editing Technologies in Kenya,11/10/2020 13. "The importance of innovation to the future of beef production", Wagyu Virtual International Conference, South Africa 11/11/2020 14. "Genome editing in livestock", National Center for genome editing in agriculture, Israel, 1/14/2021 15. "Genome editing approaches to augment cattle breeding programs", British Cattle Breeders Club Virtual Conference, England 1/26/2021 16. "How Genes Advance Progress: Genomics' Role in Beef Sustainability?" Global Roundtable for Sustainable Beef virtual webinar, 2/26/2021 17. "Biotechnology, Genomics, and Reproduction: The Ultimate Ménage à Trois" Spring 2021 RPBO Seminar Series (Warnick Lecture), University of Florida, FL 3/10/2021 18. "Gene Editing in Livestock: What is Gene Editing?" UC Davis Virtual Picnic Day, 4/17/2021 19. "Genome Editing in Animals", Invited virtual lecture to "GMO & Biosafety" course, Brac University, Bangladesh 5/5/2021 20. "Advanced Breeding Techniques Using Genomics, Reproduction, & Biotechnology" Invited virtual lecture to Cornell University, Faculty of Animal Science, Ithaca, NY 5/11/2021 21. "Regulation of Gene Editing in Livestock", Society for In Vitro Biology's Virtual Annual Meeting, SIVB 2021: In Vitro OnLine 6/9/2021 22. "Gene Editing Today and in the Future", Beef Improvement Federation Annual Research Symposium and Convention, Des Moines, Iowa 6/24/2021 What do you plan to do during the next reporting period to accomplish the goals?In the next year we plan to develop a novel approach to introduce targeted knock-ins into electroporated zygotes from four important food animal species (cattle, sheep, goats, pigs) and finish optimizing the conditions for the electroporation of goat zygotes.
Impacts
What was accomplished under these goals?
We were able to optimize electroporation conditions for bovine, porcine and ovine zygotes for the efficient (>95% knockout) generation of genome-edited H11 knockout blastocysts. We are now working on optimizing conditions for caprine zygotes. We also developed a protocol to produce in vitro fertilized sheep zygotes. This information will enable the streamlining of genome editing in livestock. The COVID-19 pandemic considerably hampered our progress as we were unable to be in the laboratory in person for most of 2020, and bench research was effectively halted for many months.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Lin, J.C. and A. L. Van Eenennaam. 2021. Electroporation-mediated genome editing of mammalian zygotes. Frontiers in Genetics.12:56 https://doi.org/10.3389/fgene.2021.648482
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Van Eenennaam, A. L. 2021. Gene editing: Today and in the Future. 2021 Proceedings of the 53rd Beef Improvement Federation Symposium and Convention. Des Moines, IA June 22-25, 2021. Pages 33-38.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Van Eenennaam, A. L. 2021. Genome editing approaches to augment cattle breeding programs. Proceedings paper in Digest 76 of the British Cattle Breeders Club. Pages 22-26.
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