Source: UNIVERSITY OF CALIFORNIA, DAVIS submitted to
GENETIC CONTAINMENT IN LIVESTOCK VIA CRISPR-MEDIATED GENE KNOCK-IN
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
EXTENDED
Funding Source
Reporting Frequency
Annual
Accession No.
1007279
Grant No.
2015-33522-24106
Project No.
CA-D-ASC-2308-CG
Proposal No.
2015-06481
Multistate No.
(N/A)
Program Code
HX
Project Start Date
Sep 1, 2015
Project End Date
Aug 31, 2020
Grant Year
2015
Project Director
Van Eenennaam, A.
Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
Animal Science
Non Technical Summary
The overall goals of this project are to advance current knowledge of gene editing tools to develop a cisgenic sterility method for genetic containment, and to concomitantly develop an approach to improve the efficiency of beef production. The idea behind the project is to try to use the SRY gene , which results in maleness, as a way to create visually-appearing males from genotypic XX females. This would be advantageous in beef cattle production where females are less efficient during the finishing phase as compared to males. This project has the potential to increase economic returns to rural communities in two ways. The first is that the development of an approach to develop all-male feedlot cattle using XSRYY bulls would improve the efficiency of beef production over the production of 50% male: 50% female offspring. Heifers are undesirable as slaughter animals as they present problems due to pregnancy and estrus, finish at lighter weights, gain weight more slowly and less efficiently than steers, and must be managed differently from steers. Obviously, this approach would only be used in terminal beef sires and not for systems looking to develop replacement heifers. Secondly, this project explores the development of an approach to contain transgenes through sterility, which may facilitate coexistence and potentially provide an acceptable cisgenic containment approach to facilitate the use of genetic engineering in animal agriculture. Additionally, information about the efficiency of CRISPR/Cas9 mediated gene insertion events and the frequency of homology directed repair as compared to non-homologous end joining when using this gene editing system will be determined. This exploratory research relates to a federal regulatory need. This project fits the stated interests of BRAG program including novel research that is not already being conducted in well-developed areas of study, and the development of approaches for co-existence. Finally this grant meets the standards of a center of excellence in large animal transgenic research, and offers an approach to produce all-male animals which could improve the efficiency and economic returns of beef cattle production. Finally, extension and public outreach materials about gene editing technologies and animal biotechnology will be developed.
Animal Health Component
0%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3013310108050%
3033310108150%
Goals / Objectives
The overall goals of this project are to advance current knowledge of gene editing tools to develop a method for genetic containment of genetically engineered animals by producing sterile animals while at the same time improving the efficiency of beef production.The four objectives of this project are to:1) Develop an efficient gene knock-in approach using the CRISPR/Cas9 system to generate cisgenic bulls carrying an extra copy of the endogenous bovine SRY gene in the non-pseudoautosomal region of the X chromosome (XSRY).2) Evaluate the effects of copy number variation in SRY on fertility of XSRYY bulls.3) Determine if this single gene knock-in of the endogenous bovine SRY gene (XSRY) is sufficient and necessary to produce all male (½ XY and ½ XXSRY) offspring from XSRYY bulls.4) Develop extension and public outreach materials that will provide information about gene editing technologies and animal biotechnology.
Project Methods
The long-term goals of this project are to advance current biotechnology tools to develop a cisgenic system of biological genetic containment through sterility in cattle, and to concomitantly develop an approach to improve the efficiency of beef production.The four objectives of this project are to:1) Develop an efficient gene knock-in approach using the CRISPR/Cas9 system to generate cisgenic bulls carrying an extra copy of the endogenous bovine SRY gene in the non-pseudoautosomal region of the X chromosome (XSRY).2) Evaluate the effects of copy number variation in SRY on fertility of XSRYY bulls.3) Determine if this single gene knock-in of the endogenous bovine SRY gene (XSRY) is sufficient and necessary to produce all male (½ XY and ½XXSRY) offspring from XSRYY bulls.4) Develop extension and public outreach materials that will provide information about gene editing technologies and animal biotechnology.It is hypothesized that CRISPR/Cas9-mediated gene knock-in of SRY will generate fertile XSRYY cisgenic bulls that, when mated with XX females, will produce phenotypically all male offspring, half of which will be non-transgenic fertile XY males, and the other half of which will be infertile cisgenic XXSRY phenotypic males, thereby providing an approach for containment of transgenes that could be transferred alongside the XSRY gene knock-in.

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

Outputs
Target Audience:The target audience includes the scientific community, livestock producers, commercial breeding companies, the global regulatory community and the general public. At this stage in the research, outreach activities have been directed to the research and regulatory communities. The target audience includes the scientific community, livestock producers, commercial breeding companies, the global regulatory community and the general public. At this stage in the research, outreach activities have been directed to the research and regulatory communities. Changes/Problems:Previously we attempted to transfer embryos after analysis using a whole genome amplification approach of biopsies taken from embryos followed by vitrification. However, each of the confirmed pregnancies was later lost. We hypothesize that the loss was due to targeting on the X chromosome site resulting in embryonic lethality, or the large number of manipulations, including injection of MII oocytes prior to fertilization, taken biopsies of the embryos and vitrification resulting in poor embryo quality. To address these concerns we selected only embryo recipients that have shown reproductive capabilities, i.e. have given birth to a calf, and addressed multiple issues associated with the quality of embryo production in the lab. Finally, we addressed the issues concerning embryo manipulations by first moving the target location to the safe harbor locus, H11, on chromosome 17 to avoid concerns with unforeseen disruption of normal gene function. Additionally, we created a donor with the green fluorescent protein under a strong promoter to identify SRY-positive embryos using fluorescent microscopy. This allows us to positively identify embryos with the correct SRY knock-in for transfer of fresh day 7 embryos to synchronized recipients without the use of biopsies or vitrification. We hope to have one calf born 3/30/2020; and possibly more from the October 25, 2019 transfer - calves would be due in July 2020. What opportunities for training and professional development has the project provided?This project is the Ph.D. project of Joseph Owen who has been working on developing the gene editing tools. He is supported on this project 100% time. How have the results been disseminated to communities of interest?"Innovation in Agricultural Science", University of Idaho, Moscow, ID "What's new in Genomics", Dairy Veterinary Services, Inc., Escalon, CA "Gene Editing in Cattle", UCCE Livestock Advisor Tour, Coalinga, CA "The possibilities and possible controversies surrounding the use of advanced reproductive technologies and genomics in animal breeding", Zoetis, Kalamazoo, MI "Food Technology: Fear or Freedom/The Need for Innovation in Agricultural Sciences", West Virginia University, Morgantown, WV, "Genome Editing Approaches for Livestock -- Advancing Animal Health and Welfare" at the Innovation in Agriculture: Women Pioneers at the Frontiers of Science, European Parliament, Brussels, Belgium "Animal Genetics and Gene Editing", K.A. Emanuel Hiel High School, Brussels, Belgium, "Gene Editing in Agriculture: Its promises and Prospects", Round table with Portuguese Government officials, Lisbon, Portugal "Gene Editing in Agriculture: Its promises and Prospects", ITQB Oeiras, Portugal "Gene Editing in Agriculture: Its promises and Prospects", Round table with Spanish Government officials, Madrid, Spain "The promise of combining advanced reproductive technologies and genomics in cattle breeding programs", University of Tennessee, Knoxville, TN "What's new in beef cattle genetics?" UCCE Beef and Range Meeting Santa Barbara County, Santa Maria, CA "What's new in beef cattle genetics?" UCCE Beef and Range Meeting Ventura County, Santa Paula, CA "Gene Editing and the Future of Bovine Genetics" 15th International Symposium on Milk Genomics & Human Health, Sacramento, CA "Public Perception of Animal Biotechnology", and "Enabling Innovation in Agricultural Breeding Programs: Promises and Prospects", Presentation to the Ugandan Ministry of Science, Technology and Innovation, Kampala, Uganda "Gene Editing in Cattle: Recent developments and prospects", Breeding for Efficiency - 2018 Efficient Dairy Genome Project Symposium, Guelph, Ontario, Canada "Were Those the Days? Animal Ag Then & Now" Iowa Pork Producers Association Annual Meeting, Des Moines, IA "Communicating Science in a Networked World" AARES Pre-conference Workshop on Evidence-based Farm and Food Policy in an Era of Fake News University of Melbourne, Victoria, Australia "Food Animal Species: The Importance of Product Risk-based Regulation of Breeding" in the Session on Gene Edited Livestock and Regulatory Stalemates. American Association for the Advancement of Science (AAAS) Annual Meeting, Washington DC, "The Role of Genetic Improvement on the Sustainability of Animal Agriculture" 2019 Midwest Veterinary Conference, Columbus, OH "Understanding Genomics, Genetic Engineering, and Gene Editing" 2019 Midwest Veterinary Conference, Columbus, OH "Veterinarian's role in Future Genetic Improvement Programs" 2019 Midwest Veterinary Conference, Columbus, OH "Dairy - where biology meets social science", 2019 Western Dairy Management Conference, Reno, NV "Genetic Improvement of Beef Cattle: Current Practice and Future Prospects", 2019 UCCE Southern San Joaquin Livestock Symposium, Cathey's Valley, CA "Genetic Improvement of Beef Cattle: Current Practice and Future Prospects", 2019 UCCE Southern San Joaquin Livestock Symposium, Visalia, CA "Genetic Improvement of Beef Cattle: Current Practice and Future Prospects", 2019 UCCE Southern San Joaquin Livestock Symposium, Bakersfield, CA "Were Those the Days? Animal Agriculture Then and Now", Northeast Agribusiness and Feed Alliance 2019 Annual Meeting and Forum, Orlando. FL Genetic Improvement of Beef Cattle: Current Practice and Future Prospects", 2019 UCCE Southern San Joaquin Livestock Symposium, Oakdale, CA "The importance of genetic innovations to sustainability", The Next New Food Panel, 42nd Annual National Food Policy Conference Consumer Federation of America Conference, Washington DC, "Gene Editing: Designing the Farm Animals and Fish of Tomorrow" Panel, Animal AgTech Innovation Summit, San Francisco, CA "A Deep Dive into Next Generation Breeding Technologies" Panel, World Agri-Tech Innovation Summit, San Francisco, CA "Genetic Improvement of Beef Cattle: Current Practice and Future Prospects", 2019 March Cattlemen's Meeting - Placer County, Lincoln, CA "Gene Editing in Agrifood: Its promises and Prospects" The Information Technology and Innovation Foundation (ITIF) Hill Class: Tech Policy 202. March 21. Internet presentation "Genome editing approaches to augmenting livestock breeding programs", JEB symposium on 'Genome editing for comparative physiology' Massa Marittima, Italy "Gene Editing in Food Animals: Prospects and Policy" French Academy of Agriculture, Paris, France "Gene Editing in Cattle: Recent developments and prospects" Invited Lecture, INRA, Jouy-en-Josas, France "Risk Assessment vs Precautionary Principle, Putting Hazard and Perception in their Proper Place for GE Organism Assessments" The International Society for Biosafety Research Conference, Tarragona Spain, "Enabling Innovation in Agricultural Breeding Programs: Promises and Prospects" The International Society for Biosafety Research Conference, Tarragona Spain, "Genetics, genomics and transgenesis: Impact in animal and food production" How biotechnology can vanish world hunger Workshop: Great challenges to Science Pirassununga, SP, Brazil, "Gene Editing in Cattle: Recent developments and prospects", Berkeley Innovative Genomics Institute, "Gene Editing in Cattle: Recent developments and prospects", Berkeley Innovative Genomics Institute Lecture, Berkeley, CA "Genetic Improvement in Food Animals: Prospects, Policy, and Public Engagement ", University and Industry Consortium, Madison, WI "Does the Animal Drug Rubric Make Sense?" University and Industry Consortium, Madison, WI "Enabling Innovation in Agricultural Breeding Programs: Promises and Prospects", UC Davis College of Agriculture and Environmental Sciences (CAES) Dean's Advisory Council Meeting, Davis, CA "Regulation of genome editing in animal agriclture: Promises and Policy", PBS (Program for Biosafety Systems), Intenet video conference to Kenya, "Enabling Innovation in Agricultural Breeding Programs: Promises and Policy", One Health Symposium, University of Delaware, DE "US Roundtable for Sustainable Beef panel", Fresno, CA "Status of Genome Editing in Food Animals", National Coalition Food and Agricultural Research (NCFAR) Lunch and Learn Session to both Senate and House Staff, Washington DC, "Gene editing for Polled", Nuffield Council on Bioethics fact-finding meeting, Internet conference, "CRISPR and Genetics and Welfare", Dairy Cattle Welfare Symposium, Orlando, FL "Comparative Evaluation of the Phenotype, Genome and Animal Products Derived from Offspring of a Genome Edited, Hornless Bull and Controls", USDA Biotechnology Risk Assessment Project Directors' Meeting, Washington DC, "The Social Challenge - Communicating the role of technology to the public" Western Section of American Society of Animal Science, Boise, ID "Genome Editing in Agriculture: Prospects and Potential Pitfalls"Ospraie Management Biennial Symposium, Invited Keynote speaker, Deer Valley, UT "Update on Gene Editing in Cattle" Invited Plenary Speaker, Beef Improvement Federation Annual Research Symposium and Convention, Brookings, SD "Overview of Gene Editing Technology", National Pork Producers Council Gene Editing Media Teleconference, Washington DC, "Importance of Innovation to Agricultural Sustainability" Invited Keynote Speaker, University of Guelph Agri-Food Excellence Symposium Guelph, Canada "The importance of genetic improvement to the sustainability of animal agriculture", Invited Awardee Presentation, Rockefeller Prentice Award in Animal Breeding and Genetics, ASAS Annual meeting, Austin, TX Owen, J.R. "Harnessing endogenous repair mechanisms for targeted gene knock-in during early bovine embryonic development", 12th biennial Transgenic Animal Research Conference, Lake Tahoe, California, What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we hope to have successfully pregnancies and live births! We plan to continue optimizing the knock-in approach via direct cytoplasmic injection to obtain the highest knock-in efficiency while also increasing developmental capabilities. Additionally, injections and embryo transfers will continue as needed until 3 confirmed pregnancies are established. Once the calves are born, whole-genome sequencing, karyotyping and fluorescent in situ hybridization will be performed to analyze the effects of the CRISPR-mediated knock-in of SRY on both male and female calves.

Impacts
What was accomplished under these goals? Objective 1: Develop an efficient gene knock-in approach using the CRISPR/Cas9 system to generate cisgenic bulls carrying an extra copy of the endogenous bovine SRY gene in the non-pseudoautosomal region of the X chromosome (XSRY). (2015-2019) - 90% completed? Progress: During this reporting period, the primary focus has been to continue to develop and optimize an efficient gene knock-in approach using the CRISPR/Cas9 system in bovine embryos, as well as a bovine fibroblast cell line. Embryo transfers of SRY-positive embryos were conducted to establish confirmed pregnancies of XSRYY embryos. Outcomes: Pregnancy rates from the embryo transfer during the previous reporting period were determined using ultrasonography. Initially, one of the recipients was shown to be pregnant but subsequently lost the pregnancy. Following this, we continued to perform experiments to establish a pregnancy from at least three XSRYY confirmed embryos. In order to establish a pregnancy, we altered our approach. After injecting embryos, we allowed them to develop in culture until day 7. On day 8, biopsies were taken and lysed. The embryos were then vitrified and stored in liquid nitrogen. The biopsies were analyzed using whole-genome amplification and the polymerase chain reaction to determine knock-in of the vitrified embryos. Once at least ten confirmed embryos with SRY inserted into the target location were obtained, recipients were estrus-synchronized for embryo transfer using standard protocols. On the day of transfer, 12 SRY-positive vitrified embryos were thawed and non-surgically transferred to recipients along with six unknown day 8 injected embryos. Pregnancies rates were then determined using ultrasonography. Three of the 18 recipients showed signs of being pregnant, while two showed signs of lost pregnancies. Following up on pregnancy checks a week later showed all five pregnancies were lost. All remaining blastocystss were analyzed and compared to biopsy results showing an overall mutation rate at the target cut-site of 82.0% and a knock-in efficiency of 34.0%. Additionally, 38.8% of knock-in embryos were non-mosaic. Due to the pregnancy losses, we developed a new approach for establishing a knock-in SRY animal. Using our previous approach, we developed a donor vector containing the green fluorescent protein (GFP) coding sequence, along with a strong eukaryotic promoter to drive GFP expression. This allows us to visualize knock-in at the target site rather than taking biopsies of the embryos at day 8. Additionally, due to the less than ideal assembly and annotation of the bovine X-chromosome, we moved target loci to the safe harbor loci, H11, on chromosome 17. H11 is a locus in mammalian genomes that resides between to non-essential genes. Previous studies have shown insertion of transgenes into the H11 locus do not disrupt normal gene function and result in a healthy functioning animal. Guides were developed targeting the bovine H11 locus and resulted in a mutation rate of 92.3% when injecting alongside Cas9 protein. The SRY-GFP donor vector was injected alongside the guide and Cas9 protein resulting in a similar knock-in efficiency to that without the GFP expression cassette. Embryos expressing GFP were whole-genome amplified and analyzed via PCR confirming insert of SRY-GFP into the H11 locus. Using this new approach, recipient cows were estrus-synchronized for embryo transfer using standard protocols on June 12th, 2019. Embryos were injected June 21th, 2019 as described and analyzed using fluorescent microscopy at day 7. Nine of the 22 embryos that reached the blastocyst stage at day 7 were positive for GFP expression. These nine embryos were loaded into embryo transfer straws containing embryo culture media and non-surgically transferred to synchronized recipients. A second round of embryos transfers were scheduled, and recipient cows were estrus-synchronized on July 3rd, 2019. Embryos were injected using the previously described approached on July 11th, 2019. The development of this second collection for embryo transfer was lower than the previous resulting in only three of the 14 embryos being GFP-positive at day 7. These three embryos were then transferred to synchronized recipients. On July 26th, 2019, blood was drawn from recipients of the June 28th transfer for pregnancy testing. One of the nine recipients showed a positive pregnancy test. On August 9th, 2019, blood was drawn from the three recipients from the July 19th transfer. Pregnancy checks were also performed at this time using ultrasonography of recipients from the June 28th transfer. These results confirmed previous pregnancy test results with one positive pregnancy. Blood results from recipients of the July 19th transfer were negative for pregnancy. These results were confirmed by ultrasonography the following week. Approximately 68 days into the pregnancy, the sex of the fetus was determined using ultrasonography showing a male phenotype. We have continued to monitor the pregnancy using ultrasonography. As of September 30th, the fetus is healthy and continuing to develop and is due March 30, 2020. Given this success, we have scheduled another round of embryo transfers for October 25th, 2019. We are hopeful that using this approach we will obtain at least one male (H11SRYXY) and one female (H11SRYXX) calf, which we predict will be phenotypically male. Objective 2: Evaluate the effects of copy number variation in SRY on fertility of H11SRYY bulls. (2020) - 0% completed? If pregnancies are confirmed at day 30, the H11SRYXY and H11SRYXX calves should be born around July 2020. We will then be able to start evaluating these animals for phenotype, off-target effects, karyotype and correct insertion of SRY into the H11 locus on the chromosome 17. Objective 3: Determine if this single gene knock-in of the endogenous bovine SRY gene (H11SRY) is sufficient and necessary to produce the male phenotype (2020) - 0% completed We ended up transferring both male and female embryos as we did not use sexed semen in the June/July 2019 embryo transfers - we therefore anticipate we will obtain H11SRYXY and H11SRYXX calves in July 2020. We predict the H11SRYXX calves will be phenotypically male, and infertile. Objective 4: Develop extension and public outreach materials that will provide information about gene editing technologies and animal biotechnology. (2015-2020) - 70% completed? We have been obtaining footage of the process of creating the XSRYY animals for development of outreach materials. In addition, the company Science Animated has created an animated video illustrating the process for creating these animals. Once the animals are born, an informational video will be produced about the gene editing process and methodology behind creating these animals.

Publications

  • Type: Theses/Dissertations Status: Published Year Published: 2019 Citation: Owen, J. R. 2019. Harnessing endogenous repair mechanisms for targeted gene knock-in during early bovine embryonic development. Unpublished Ph.D. Dissertation. University of California, Davis, CA.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: Owen, J.R, S.L. Henning, E.E. Paulson, J.C. Lin, A.E. Young, L.C. Cabrera, J.D. Murray, P.J. Ross, A.L. Van Eenennaam. 2019. Harnessing endogenous repair mechanisms for targeted gene knock-in during early bovine embryonic development. Transgenic Research.


Progress 09/01/17 to 08/31/18

Outputs
Target Audience:The target audience includes the scientific community, livestock producers, commercial breeding companies, the global regulatory community and the general public. At this stage in the research, outreach activities have been directed to the research and regulatory communities.The target audience includes the scientific community, livestock producers, commercial breeding companies, the global regulatory community and the general public. At this stage in the research, outreach activities have been directed to the research and regulatory communities. Changes/Problems:The incorrect annotation of the bovine genome slowed progress on this project considerably, and the May 2018 release of the updated assembly has enabled us to now correctly target the X chromosome and obtain edited embryos. These have been successfully transferred to recipient cows. We are now awaiting pregnancy status of those animals, Given our high knock-in efficiency rate of 47.8% (11/23) in bovine embryos, we anticipate no further delay and expect to have edited calves on the ground by the next reporting period. It should be noted that this knock-in rate is exceptionally high as compared to the published literature, and a paper outlining how we were able to accomplish this is forthcoming. What opportunities for training and professional development has the project provided?This project is the Ph.D. project of Joseph Owen who has been working on developing the gene editing tools. He is supported on this project 100% time. How have the results been disseminated to communities of interest?Presentations Van Eenennaam, A.L. "Innovation in Agricultural Science Lecture: Animal Genome Editing", 2017 National Association of State Departments of Agriculture (NASDA) Annual Meeting, New Orleans, LA, 9/14/2017 Van Eenennaam, A.L. "The Future of Genetic Alteration in Food Animal Production", 50th Annual Conference of the American Association of Bovine Practitioners, Omaha, NE, 9/15/2017 Van Eenennaam, A.L. "Proposed Regulation of Gene Edited Animals in the U.S.", Netherlands Commission on Genetic Modification (COGEM), Rotterdam, The Netherlands, 10/20/2017 Van Eenennaam, A.L. "Use of Gene Editing in Cattle Breeding", 2017 American Embryo Transfer Association (AETA) and Canadian Embryo Transfer Association (CETA/ACTE) Joint Convention, Orlando, FL, 10/27/2017 Van Eenennaam, A.L. "Will Genome Editing Be Embraced or Eschewed?", North Dakota State University Animal Science Department Graduate Seminar Series, Fargo, ND, 11/7/2017 Van Eenennaam, A.L. "Regulatory oversight of new breeding innovations in the U.S.", TropAgBio2017, Queensland, Australia, 11/19/2017 Van Eenennaam, A.L. "Science communication to obtain social license for use of genome editing in animal breeding programs", The Canadian Agriculture and Agri-Food Genomics Forum, Toronto, Canada, 11/30/2017 Van Eenennaam, A.L. "Innovation in Agricultural Science", 2017 Agribusiness Roundtable, Tempe, AZ, 12/4/2017 Van Eenennaam, A.L. "The Use and Impact of Genetic Technologies in the Dairy Industry", Professional Dairy Producers of Wisconsin (PDPW) Food & Policy Summit, Madison, WI, 12/7/2017 Van Eenennaam, A.L. "Innovation in Agricultural Science?", 2018 Legislative Agriculture Chairs Summit, State Agriculture and Rural Leaders & Council of State Governments, Kansas City, MO 1/6/2018 Van Eenennaam, A.L. "Gene Editing", American Farm Bureau Convention, Nashville, TN, 1/8/2018 Van Eenennaam, A.L. "Biotechnology - promise and politics", College of Life Sciences, Brigham Young University, Provo, UT, 1/18/2018 Owen, J.R. "Genetic containment in livestock via CRISPR-mediated gene knock-in", USDA BRAG PD Annual Meeting, 5/22/2018 Abstracts Van Eenennaam, A.L. "The Future of Genetic Alteration in Food Animal Production", 50th Annual Conference of the American Association of Bovine Practitioners, Omaha, NE, 9/15/2017 Van Eenennaam, A.L. "Proposed Regulation of Gene Edited Animals in the U.S.", Netherlands Commission on Genetic Modification (COGEM), Rotterdam, The Netherlands, 10/20/2017 Van Eenennaam, A.L. "Regulatory oversight of new breeding innovations in the U.S.", TropAgBio2017, Queensland, Australia, 11/19/2017 Van Eenennaam, A.L. "Science communication to obtain social license for use of genome editing in animal breeding programs", The Canadian Agriculture and Agri-Food Genomics Forum, Toronto, Canada, 11/30/2017 Owen, J.R. "Genetic containment in livestock via CRISPR-mediated gene knock-in", The Society for the Study of Reproduction Annual Meeting, 7/13/2018 Proceedings Van Eenennaam, A.L. "The Future of Genetic Alteration in Food Animal Production", 50th Annual Conference of the American Association of Bovine Practitioners, Omaha, NE, 9/15/2017 Press "Making happier animals? Gene editing on the farmyard", Science Friction (ABC radio Australia), 11/17/2017 http://radio.abc.net.au/programitem/perQlnEV5D?play=true "Cattle Geneticist and Active Spokesperson for Ag", Successful Farming at Agriculture.com, 11/23/2017, https://www.agriculture.com/livestock/cattle/cattle-geneticist-and-active-spokesperson-for-ag "Geneticist Alison Van Eenennaam: Genetic engineering could save farm animals from disease", Genetic Literacy Project, 11/29/2017, https://geneticliteracyproject.org/2017/11/29/meet-animal-geneticist-alison-van-eenennaam-one-agricultures-leading-voices-reason/? What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we plan to continue optimizing the knock-in approach via direct cytoplasmic injection in order to obtain the highest knock-in efficiency possible. Additionally, injections and embryo transfers will continue as needed until 3 confirmed pregnancies are established. Once the calves are born, whole-genome sequencing, karyotyping and fluorescent in situ hybridization will be performed to analyze the effects of the CRISPR-mediated knock-in of SRY on the bull calves.

Impacts
What was accomplished under these goals? Objective 1: Develop an efficient gene knock-in approach using the CRISPR/Cas9 system to generate cisgenic bulls carrying an extra copy of the endogenous bovine SRY gene in the non-pseudoautosomal region of the X chromosome (XSRY). (2015-2018) - 90% completed Progress: During this reporting period, the primary focus has been to continue to develop and optimize an efficient gene knock-in approach using the CRISPR/Cas9 system in bovine embryos, as well as a bovine fibroblast cell line. Outcomes: Experiments were conducted to optimize conditions with respect to injection of Cas9 mRNA, protein, and a combination of the two. A plasmid was constructed for use as the donor template to knock the SRY gene into the target locus and injected into single cell bovine zygotes alongside the gRNA and Cas9 protein, as well as transfected into our bovine fibroblast line. This has led to successful insertion of SRY at the target locus in both bovine embryos and our bovine fibroblast line using whole genome amplification in bovine embryos. However, with integration of SRY at the target loci, we were unable to establish an isogenic knock-in cell line for somatic cell nuclear transfer cloning. With the recent release (May 2018) of the greatly improved bovine genome sequence, it was revealed that the target locus region was improperly annotated on the previous genome sequence, resulting in cell death when inserting the gene at this location. Following this discovery, new guides were created targeting a region 10kb downstream of the target gene. Oligos were ordered and the new guides were in vitro transcribed for testing via direct injection of bovine embryos. Of the four guides, one guide resulted in a greater than 80% mutation rate at the target site when injected alongside Cas9 protein. A new donor vector was created surrounding the new cut site and assembled using Gibson assembly along with the previous approach. The gRNA, Cas9 protein and donor vector were injected into in vitro fertilized embryos and cultured to the blastocyst stage. Embryos were analyzed using whole genome amplification and PCR, showing successful knock-in of SRY at the new target locus. This approach was repeated over multiple collections, resulting in an overall mutation rate at the target of 86.1% and an overall knock-in rate of 28.1%. Recipient cows were estrus-synchronized for embryo transfer using standard protocols. Embryos were injected as described and biopsies were taken at day eight. Re-expanded embryos were then non-surgically transferred to recipients. Three out of the ten (30%) embryos (2 female and 1 male) were positive for SRY knock-in at the target locus. Embryos that were not transferred were collected and underwent whole-genome amplification. The target locus was analyzed for knock-in efficiency using PCR, showing a knock-in efficiency of 47.8% (11/23). Pregnancy rate will be determined using ultrasonography during the week of November 17th, 2018 To ensure successful pregnancy of an XSRYY male, oocytes will be injected, biopsies will be taken and blastocysts will be vitrified over the next four weeks. Recipients will then be synchronized and male embryos positive for SRY knock-in will be non-surgically transferred at the beginning of November 2018. Objective 2: Evaluate the effects of copy number variation in SRY on fertility of XSRYY bulls. (2019-2020) - 0% completed If pregnancies are confirmed at day 30, the XSRYY bull calves should be born around late June, 2019. We will then be able to start evaluating these animals for off-target effects, proper karyotype and correct insertion of SRY into the ZFX locus on the X-chromosome. Once they reach sexual maturity in September of 2020 we can evaluate the breading soundness of the bull calves. Objective 3: Determine if this single gene knock-in of the endogenous bovine SRY gene (XSRY) is sufficient and necessary to produce all male (1⁄2 XY and 1⁄2 XXSRY) offspring from XSRYY bulls. (2020) - 0% completed Once the XSRYY bulls reach sexual maturity, we will mate the bulls to non-cisgenic females. The offspring will be collected at day 90 and phenotypic sex will be determined. Karyotyping and FISH will be performed to determine genotypic sex and genomic position of the SRY gene. Objective 4: Develop extension and public outreach materials that will provide information about gene editing technologies and animal biotechnology. (2015-2020) - 40% completed We have been obtaining footage of the process of creating the XSRYY animals for development of outreach materials. Once the animals are born, an informational video will be produced about the gene editing process and methodology behind creating these animals.

Publications

  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Van Eenennaam, A.L. Will Genome Editing Be Embraced or Eschewed?, North Dakota State University Animal Science Department Graduate Seminar Series, Fargo, ND, 11/7/2017
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Van Eenennaam, A.L. Regulatory oversight of new breeding innovations in the U.S., TropAgBio2017, Queensland, Australia, 11/19/2017
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Van Eenennaam, A.L. Science communication to obtain social license for use of genome editing in animal breeding programs, The Canadian Agriculture and Agri-Food Genomics Forum, Toronto, Canada, 11/30/2017
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Van Eenennaam, A.L. Innovation in Agricultural Science, 2017 Agribusiness Roundtable, Tempe, AZ, 12/4/2017
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Van Eenennaam, A.L. The Use and Impact of Genetic Technologies in the Dairy Industry, Professional Dairy Producers of Wisconsin (PDPW) Food & Policy Summit, Madison, WI, 12/7/2017
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Van Eenennaam, A.L. Innovation in Agricultural Science Lecture: Animal Genome Editing, 2017 National Association of State Departments of Agriculture (NASDA) Annual Meeting, New Orleans, LA, 9/14/2017
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Van Eenennaam, A.L. The Future of Genetic Alteration in Food Animal Production, 50th Annual Conference of the American Association of Bovine Practitioners, Omaha, NE, 9/15/2017
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Van Eenennaam, A.L. Proposed Regulation of Gene Edited Animals in the U.S., Netherlands Commission on Genetic Modification (COGEM), Rotterdam, The Netherlands, 10/20/2017
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Van Eenennaam, A.L. Use of Gene Editing in Cattle Breeding, 2017 American Embryo Transfer Association (AETA) and Canadian Embryo Transfer Association (CETA/ACTE) Joint Convention, Orlando, FL, 10/27/2017
  • Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Van Eenennaam, A.L. Innovation in Agricultural Science?, 2018 Legislative Agriculture Chairs Summit, State Agriculture and Rural Leaders & Council of State Governments, Kansas City, MO 1/6/2018
  • Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Van Eenennaam, A.L. Gene Editing, American Farm Bureau Convention, Nashville, TN, 1/8/2018
  • Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Van Eenennaam, A.L. Biotechnology  promise and politics, College of Life Sciences, Brigham Young University, Provo, UT, 1/18/2018
  • Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Owen, J.R. Genetic containment in livestock via CRISPR-mediated gene knock-in, USDA BRAG PD Annual Meeting, 5/22/2018
  • Type: Other Status: Other Year Published: 2018 Citation: Owen, J.R. Genetic containment in livestock via CRISPR-mediated gene knock-in, The Society for the Study of Reproduction Annual Meeting, 7/13/2018
  • Type: Other Status: Other Year Published: 2017 Citation: Making happier animals? Gene editing on the farmyard, Science Friction (ABC radio Australia), 11/17/2017 http://radio.abc.net.au/programitem/perQlnEV5D?play=true
  • Type: Other Status: Other Year Published: 2017 Citation: Cattle Geneticist and Active Spokesperson for Ag, Successful Farming at Agriculture.com, 11/23/2017, https://www.agriculture.com/livestock/cattle/cattle-geneticist-and-active-spokesperson-for-ag
  • Type: Other Status: Other Year Published: 2017 Citation: Geneticist Alison Van Eenennaam: Genetic engineering could save farm animals from disease, Genetic Literacy Project, 11/29/2017, https://geneticliteracyproject.org/2017/11/29/meet-animal-geneticist-alison-van-eenennaam-one-agricultures-leading-voices-reason/


Progress 09/01/16 to 08/31/17

Outputs
Target Audience:The goal of this project is to use the SRY gene, which results in maleness, as a way to create all-male offspring. This approach could be used to contain transgenes to a single generation since the resulting offspring would be infertile and would be advantageous for beef cattle production since females generally produce less beef than males. This project has the potential to increase economic returns to rural communities in two ways. Firstly, the development of an approach to develop all males would improve the efficiency of beef production over current production methods, which produce half males and half females, on average. Heifers are less desirable as slaughter animals as they present challenges due to pregnancy and estrus, finish at lighter weights, gain weight more slowly and less efficiently than steers, and must be managed differently from steers. Secondly, this project explores the development of an approach to contain transgenes through sterility, which may facilitate coexistence and potentially provide an acceptable biological containment approach to facilitate the use of genetic engineering in animal agriculture. This project will provide data on the use of new gene editing techniques, particularly CRISPR/Cas9, in cattle. It will also provide information that can be used to inform the federal regulatory process with respect to gene-edited animals. This project fits the stated interests of the BRAG program including novel research that is not already being conducted in well-developed areas of study, and the development of approaches for co-existence. Finally, this grant meets the standards of a center of excellence in large animal transgenic research, and offers an approach to produce all-male animals, which could improve the efficiency and economic returns of beef cattle production. Extension and public outreach materials about gene editing technologies and animal biotechnology will be developed in order to make all information produced by these efforts accessible to the general public. The target audience includes the scientific community, livestock producers, commercial breeding companies, the global regulatory community and the general public. At this stage we are still in the research stage and so outreach activities have been directed to the research and regulatory communities. Changes/Problems:Despite repeated attempts, we have experienced problems obtaining a homologous recombination of the bovine SRY gene in embryos. We are therefore shifting our focus to try to accomplish a knock-in in a male fibroblast culture line followed by somatic cell nuclear transfer (SCNT) cloning to obtain the XSRYY bulls. The advantage of this approach is that we will be working with a confirmed knock-in in a male cell line prior to cloning. The disadvantage is the known inefficiencies associated with cloning. We have established 3 different fibroblast lines to try to maximize the probability that we will successfully obtain a knock-in line that can be successfully used in SCNT. This has delayed the accomplishment of Goal 1. This delay in successfully obtaining a gene knock-in will likely result in the requirement of a one year no-cost extension for this project due to the length of bovine gestation and sexual maturity. What opportunities for training and professional development has the project provided?This project is the Ph.D. project of Joseph Owen who has been working on developing the gene editing tools. He is supported on this project 100% time. How have the results been disseminated to communities of interest?35 presentations given during the past year: "Gene Editing: Breeding or GMO?", Genome Editing and the Future of Farming, Edinburgh, Scotland 9/6/2016 "Animal Biotechnology: Opportunities and Challenges", Cornell Alliance for Science Fellows Training Program, Ithaca, NY 9/13/2016 "Gene editing: what is it and how might it be used in animal breeding programs", American Wagyu Association Conference, Cour d'Alene, ID 9/15/2016 "The #SciComm challenge facing animal agriculture", 2016 Allen D. Leman Swine Conference, St. Paul, MN 9/20/2016 "Animal Health and Wellbeing", BIO Animal Biotech Summit, Washington, DC 9/21/2016 "Regulation Challenges and Tactics", Biotech University, Saskatoon, Saskatchewan, Canada 9/29/2016 "The #SciComm Challenge Facing Agriculture Technologies", Biotech University, Saskatoon, Saskatchewan, Canada 9/29/2016 "Gene editing: Breeding or Genetic Engineering?", Biotech University, Saskatoon, Saskatchewan, Canada 9/30/2016 "The synergistic use of molecular markers, biotechnology, genomic selection and advanced reproductive technologies in livestock breeding programs", XVI Latin American Genetics Congress, Montevideo, Uruguay 10/10/2016 "Genome Editing: What's all the fuss?", NBCEC Brown Bagger Series, webinar 10/12/2016 "Genome Editing", National Association of Animal Breeders (NAAB) 70th Annual Convention, Green Bay, WI 10/14/2016 "Animal Biotechnology: What is, what could be, and will it be?", Marian Koshland Memorial Lecture, Berkeley, CA 11/29/2016 "Genomics 101 and potential uses of gene editing in the California Beef Industry", California Cattlemen's Association meeting, Reno, NV 12/1/2016 "The synergistic use of molecular markers, biotechnology, genomic selection and advanced reproductive technologies in livestock breeding programs", UC Davis Veterinary School Food Animal Reproduction and Medicine Club, Davis, CA 1/7/2017 "Gene Editing - The Pros and Cons and Relevance to the Beef Sector", British Cattle Breeder's Club, Telford, Shropshire, England 1/24/2017 "Animal Biotechnology: What is it, what could it be, and will it be allowed?", York Distinguished Lecture, Auburn University 2/14/2017 "How Genome Editing Can Synergistically Accelerate Animal Genetic Improvement Programs", American Association for the Advancement of Science (AAAS) 2017 Annual Meeting, Boston, MA "Advanced Genetic Technologies", Snyder Livestock Co. Bull Test Sale, Yerinton, NV 3/11/2017 "Will gene editing face the same fate as genetic engineering? The #scicomm challenge", AgriBio, LaTrobe University, Bundoora, Victoria, Australia 3/21/2017 "The #SciComm Challenge Facing Agriculture", Hunter College of the City University of New York, New York, NY 3/31/2017 "Impacts of the Revolutionary Technology (CRISPR & other biotech) on Crop & Livestock Agriculture and Bio-Engineering Value Chains", Strategic Perspectives on Innovation in Agrifood Supply Chains: Profitability, Sustainability, and Global Change, UC Berkeley, Berkeley, CA 4/18/2017 "Regulatory environment for new technologies", The Analytical Excellence Through Industry Collaboration (AEIC) Spring Meeting 2017, Santa Clara, CA 4/19/2017 "The #Scicomm challenge facing animal agriculture", New Horizons Seminar Series, School of Veterinary Medicine, UC Davis, Davis, CA 4/26/2017 "Gene Editing in Animal Breeding", GeneSeek Science Advisory Counsel, Lincoln, NE 5/9/2017 "Will breeders be able to use genome editing in livestock improvement programs?", Impacts and Applications of Genome Editing Technologies, Michigan State University, East Lansing, MI 5/10/2017 "Impact and Applications of Genome Editing Technologies: What is the cost of giving up on technology?", Zoetis Dairy Wellness Summit, AZ 5/18/2017 "Genetic Containment of Livestock Via CRISPR-mediated Gene Knock-in", USDA Biotechnology Risk Assessment Grants Program PD Meeting, Washington, DC 5/23/2017 "Genetic Improvement of Food Animals: Past and Future", International Consortium on Applied Bioeconomy Research (ICABR), Berkeley, CA 5/31/2017 "Advancements in emerging technology: How genome editing could synergistically accelerate animal genomics", Beef Improvement Federation Conference, Athens, GA 6/2/2017 "Agriculture biotech, public policy, and media in a post-truth era", Bootcamp on Public Trust in Agricultural Technololgy, Guelph, Canada 6/6/2017 "Regulatory trends and `New Breeding Techniques': Canada, U.S., Europe", Bootcamp on Public Trust in Agricultural Technology", Guelph, Canada 6/7/2017 "How genome editing could synergistically accelerate animal genomics", Monsanto Fellow Colloquium, St. Louis, MO 6/8/2017 "Animal Biotechnology: Opportunities and Obstacles", Genetic Engineering in Agriculture - Science, Policy, and Law, Agribusiness Committee of the California State Bar Business Section, Davis, CA 6/13/2017 "Will Animal Genetics Innovations Be Embraced or Eschewed? The #Scicomm Challenge Facing Agricultural Biotechnology", International Society of Animal Genetics (ISAG), Dublin, Ireland 7/20/2017 "Science communication to obtain social license for use of genome editing in animal breeding programs", 68th Annual Meeting of the European Federation of Animal Science", Tallinn, Estonia 8/29/2017 We have also been involved in a number of media events associated with gene editing in livestock · "The Age of Genetically Engineered Animals Has Arrived", Transgenic News · "Gene editing: Improved animal welfare and food security?", Agricultural with Dr. Lindsay · "Bio-engineered news", Sacramento News and Review · "2016 BIO Animal Biotech Summit Day One Hightlights", BIOtechNOW · "Global Biotech Week in Saskatoon", CTV News Saskatoon, Saskatchewan, Canada · "Alison Van Eenennaam and Jennifer Kuzma: How should hornless cows and gene editing be regulated", Genetic Literacy Project · "Genetically Engineered Animals Could Ease World Hunger", AgWeb · "Genetic Engineering's New Age", Transgenic News · "Gene-edited animals face US regulatory crackdown", Nature News · "Will Biotechnology Regulations Squelch Food and Farming Innovation?" SEARCA Biotechnology Information Center · "Directing Nature? Gene Editing Offers Big Potential", Beef Magazine · "Gene Editing Can Complement Traditional Food-Animal Improvements", UC Davis News · "Gene editing can complement traditional food-animal improvements", Science Daily · "FDA moves to regulate gene editing as a drug", Western Livestock Journal · "If DNA is a drug, then all life on Earth is high", Western Livestock Journal · "Gene editing can complement traditional food animal improvements", Phys.org · "Gene Editing Can Complement Traditional Food-Animal Improvements", BioQuickNews · "Changes to Chickens and More", GenomeWeb · "Gene editing mulled for improving livestock", Phys.org · "Gene Editing Can Produce Hornless Cows and Boost Livestock Production - Here's How", Tech Times · "Gene editing mulled for improving livestock", The Nation · "Gene editing could help improve livestock - experts", Irish Sun, Breaking Property News, Nigeria Sun, Malaysia Sun, Germany Sun, Northern Ireland News · "Gene Editing Can Produce Hornless Cows and Boost Livestock Production - Here's How", The Bullvine · "Genome Editing Introduces Desirable Genetic Variations Into Livestock Breeding Programs", American Laboratory · "Gene Editing Livestock", Inquiring Minds · "FDA proposes to regulate all animals with `intentionally altered' DNA as drugs", ASAS Taking Stock · "CRISPR the next great Disruptor in Crops and Animal Health", Linked In · "From Corn to Cattle, Gene Editing Is About to Supercharge Agriculture", Digital Trends · "From Corn To Cattle, Gene Editing Is About To Supercharge Agriculture", interestingthingsonline.com What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we plan to continue to develop an efficient approach to performing a gene knock-in of SRY in bovine embryos. The next step in embryos is to inject the gRNA with either Cas9 mRNA or protein alongside the ssODN, a double-stranded ODN, the linearized donor plasmid and the circular donor plasmid. Additionally, recent studies have shown that incubating the embryos in Scr7, an inhibitor of NHEJ and RS-1, increases the efficiency of performing a gene knock-in using this system in mice and rabbits. We will directly inject 50μM concentrations of Scr7 and RS-1 individually, as well as the two combined, into embryos alongside the varying donor vectors and either Cas9 mRNA or protein to determine which combination results in the highest knock-in efficiency while yielding the highest success rate of embryo production. Once we have optimized this system, we will begin transferring XSRYY confirmed embryos into surrogate heifers. Additionally, a donor plasmid is currently being constructed as mentioned above for attempting a knock-in using a bovine cell line for somatic cell nuclear transfer cloning. This donor will contain the 1.8kb SRY sequence followed by the puromycin coding sequence flanked by lox-P sites. This will allow us to select for cells that have the SRY knock-in using a long course puromycin selection period, which we can then remove using Cre once the knocked-in cell line is established. After the XSRYY bull calves are born, we can begin to evaluate the effects of copy number variation in SRY on fertility of XSRYY bulls. Additionally, once the XSRYY bulls reach sexual maturity, we will mate them to heifers and analyze the offspring to determine if this single gene knock-in of the endogenous bovine SRY gene (XSRY) is sufficient and necessary to produce all male (1⁄2 XY and 1⁄2 XXSRY) offspring. Finally, we will continue to video document the steps involved in this project in order to develop extension and public outreach materials that will provide information about gene editing technologies and animal biotechnology.

Impacts
What was accomplished under these goals? The overall goal of this project is to generate cattle with an SRY gene knock-in via CRISPR technology that will enable them to produce only male offspring, which are more desirable from a production standpoint, with the added feature of containing the transgene to a single generation since those male offspring will presumably be sterile. During this reporting period, the primary focus has been to continue to develop and optimize an efficient gene knock-in approach using the CRISPR/Cas9 system in bovine embryos, as well as a bovine fibroblast cell line. Objective 1. Develop an efficient gene knock-in approach using the CRISPR/Cas9 system to generate cisgenic bulls carrying an extra copy of the endogenous bovine SRY gene in the non-pseudoautosomal region of the X chromosome (XSRY). Years 1-3 - 50% completion Methods using guide RNAs (gRNAs) that were developed and tested during the previous reporting period for targeting the knock-in locus have been optimized in both bovine embryos and cell lines. Ten in vitro transcribed (IVT) guide-RNAs were constructed using the online tool sgRNA Scorer 2.0 and tested using an in vitro cleavage assay. Two of these gRNAs showed 100% cleavage in vitro and were injected into single cell bovine zygotes alongside Cas9 mRNA, protein and a combination of mRNA and protein. The embryos were allowed to develop to the blastocyst stage, lysed and the target region was amplified using the polymerase chain reaction (PCR). Sanger sequencing showed a 57.9% mutation rate at the cut site when injecting gRNAs alongside Cas9 mRNA, an 81.7% mutation rate at the cut site when injection gRNAs alongside Cas9 protein and an 83.6% mutation rate at the cut site when injecting gRNAs alongside both Cas9 mRNA and protein. These results showed that we could obtain a high rate of cleavage at the target site by injecting gRNAs alongside Cas9 protein. After gRNA selection, a plasmid containing the 1.8kb sequence with the endogenous SRY promoter and coding sequence and 1kb homology arms homologous to the 1kb of sequence flanking either side of the gRNA cut site was constructed as the donor template to knock-in SRY at the target locus. This plasmid was injected into single cell bovine zygotes alongside the gRNA and Cas9 protein. Groups of 30 zygotes were injected and incubated with and without RS-1, a small molecule known to increase the DNA binding affinity of RAD51, a protein associated with homology directed repair (HDR). Embryos were allowed to develop and were lysed at the blastocyst stage; the target region was amplified using PCR to detect SRY knock-ins. No successful knock-ins were observed. Verification that the guides were cutting at the target region when injected alongside the donor vector was performed and showed an 84.6% (44/52) mutation rate at the cut site when incubated with RS-1 and a 71.4% (35/49) mutation rate at the cut site with no RS-1. This showed that the lack of knock-ins was not due to cleavage problems at the target site. Next, a single stranded oligo donor nucleotide (ssODN) was constructed from the plasmid donor and microinjected into single cell bovine zygotes alongside the gRNA and Cas9 protein. Groups of 30 zygotes were injected and incubated with and without RS-1. Embryos were allowed to develop and were lysed at the blastocyst stage; the target region was amplified using PCR to analyze for SRY knock-ins. No successful knock-ins of SRY were observed. Embryos injected with the ssODN and incubated with RS-1 had an 86.4% (19/22) mutation rate at the cut site and embryos injected with the donor and no RS-1 had an 84.2% (16/19) mutation rate at the cut site. Similar to injections with the donor plasmid, the lack of insertion of SRY at the target location was not due to a lack of cleavage efficiency at the target site. A male bovine fibroblast cell line from a Holstein bull was also established to attempt a knock-in of SRY. Electroporation determined a transfection efficiency of 54.7% for this cell line. To easily identify cells with successful knock-ins, a puromycin kill-curve was performed to determine the minimum inhibitory concentration (MIC) for puromycin. The gRNAs optimized using bovine embryos were inserted into a plasmid for in vivo gRNA expression in mammalian cell lines. The gRNA plasmid was electroporated alongside a Cas9-2A-puromycin expression vector and Cas9 transfected cells were selected using the puromycin MIC. DNA was extracted from electroporated cells and showed a 61.6% cleavage after puromycin selection. These results showed a high cleavage effiency at the target site using this cell line approach. This will allow us to attempt a knock-in of SRY in millions of cells rather than small groups of embryos. Once established, these cells could be used for cloning. We have opened an INAD (Investigational New Animal Drug) for this project with the FDA and have obtained a food use authorization for the surrogate dams that are going to carry the XSRYY bulls Objective 2. Evaluate the effects of copy number variation in SRY on fertility of XSRYY bulls. Year 4 - 0% completion as we have not yet obtained pregnancies Objective 3. Determine if this single gene knock-in of the endogenous bovine SRY gene (XSRY) is sufficient and necessary to produce all male (1⁄2 XY and 1⁄2 XXSRY) offspring from XSRYY bulls. Year 5 - 0% completion Objective 4. Develop extension and public outreach materials that will provide information about gene editing technologies and animal biotechnology. Year 5 - 20% completion A total of 35 presentations relating to gene editing in animals were given to audiences in many locations. Additionally, the PD participated in many media opportunities around gene editing in livestock. Objective 4 will primarily be completed towards the end of the project, we have video documented the methods and procedures involved in research surround the development of an efficient approach to performing a gene knock-in using bovine embryos.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Carroll, D., Van Eenennaam, A., Taylor, J.R., Seger, J., Voytas, D. 2016. Regulate genome-edited products, not genome editing itself. Nature Biotechnology. 34(5):477-479. https://www.nature.com/articles/nbt.3566.pdf?origin=ppub
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Petracca, Y., Van Eenennaam, A.L., Lema, M.A. 2016. Gene Editing: Do not forget about Animal Agriculture. Journal of Advanced Research in Biotechnology. 1(1):1-2.�http://www.symbiosisonlinepublishing.com/biotechnology/biotechnology09.pdf
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Genetic Modification of Food Animals. Current Opinion in Biotechnology 44:27-34. http://www.sciencedirect.com/science/article/pii/S0958166916302348
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Van Eenennaam, A. L. (2016). Gene editing: Breeding or GMO?. National Institutes of Bioscience Journal, 1. https://doi.org/https://doi.org/10.2218/natlinstbiosci.1.2016.1747


Progress 09/01/15 to 08/31/16

Outputs
Target Audience:The target audience for the research will ultimately be producers in the livestock industries. At this stage we are still in the research stage and so outreach activities have been directed to the research and regulatory community. Changes/Problems:The sole change to the project is the cell line used in specific aim 1.1. Here we changed the cell line from primary bovine fibroblast cells to Madin-Darby bovine kidney (MDBK) cells. This change is cell line was due to the low transfection efficiency via Lipofectamine and electroporation of primary fibroblast cells. It has previously been shown that primary fibroblast cell lines can be difficult to transfect (Jordan et al, 2008), which we have demonstrated by the increased transfection efficiency of eGFP via electroporation when switching from bovine fibroblast to MDBK. Jordan ET, Collins M, Rubio T, et al. 2008. Optimizing Electroporation Conditions in Primary and Other Difficult-to-Transfect Cells. J. Biomol. Tech. 19(5): 328-334. What opportunities for training and professional development has the project provided?This project is the Ph.D. project of Joseph Owen who has been working on developing the gene editing tools. He is supported on this project 100% time. How have the results been disseminated to communities of interest?Presentations have been given to the following audiences: 1. "Food for Thought: Innovations and Opportunities for Animal Breeding", Explorit Science Center lecture series, Davis, CA 9/1/15 2. "Animal Biotechnology: Opportunities and Challenges" Cornell Alliance for Science Global Fellows Science Week, Cornell, Ithaca, NY 9/9/15 3. "How Animals are Genetically Engineered" Cornell GMO Debate Course, Cornell, Ithaca, NY 9/10/15 4. "Overview of the Ethical Issues of Germ Line Modification in Animals" at The National Academies' Board on Life Sciences/Institute for Laboratory Animal Research meeting on "Workshop on Gene Editing to Modify Animal Genomes for Research - Scientific and Ethical Considerations" in Washington DC 12/8/15 5. "The use of biotechnology in animal agriculture: past, present, and future" CTNBio, Brasilia, Brazil 12/9/15 6. "Assessing the potential impacts of biotechnology: evaluating risks and benefits" CTNBio, Brasilia, Brazil 12/10/15 7. "The Use of Biotechnology in Animal Agriculture" , Seminar for 17 faculty from Jiangsu Province, China, who are attending the One Health for Food Safety Conference for Animal and Veterinary Scientists, Nov 30-Dec 18, 2015, UC Davis, CA 12/15/15 8. "Gene Editing: Breeding or Genetic Engineering?" Plant and Animal Genome XXIV. San Diego, CA 1/09/16 9. "The use of GMOs in animal agriculture" Western Canadian Association of Bovine Practitioners (WCABP), Calgary, AB, Canada 1/14/16 10. "GMOs Use In Animal Agriculture", National Cattlemen's Beef Association Cattlemen's College, San Diego, CA 1/27/16 11. "Animal Biotechnology and the Livestock Revolution" Cornell University Plant Breeding Symposium, Ithaca, NY 3/11/16 12. "The Role of Animal Biotechnology in the 21st Century" BioVision, Alexandria, Egypt 4/13/16 13. "The Current and Future Uses of Biotechnology in Animal Agriculture", Ensminger University Conference, Honduras, 5/14/16 14. "Emerging Genetic Advancements", Center for Food Integrity Conference, Hamburger University, McDonald's Campus, Chicago, IL, 5/18/16 15. "Use of GMOs in Animal Agriculture Production", Sonoma-Marin Cattlemen' s Association Field Day & BBQ, 6/5/16 16. "Animal Biotechnology", BIO 2016 FutureMakers Ted-style talk, BIO convention, San Francisco, CA 6/8/16 17. "The Future of Meat", Breakthrough Institute Dialogue, Sausalito, CA, 6/23/15 18. "Animal genomics and biotechnology in production systems", Ninth Latin American and Caribbean Agricultural and Forestry Biotechnology meeting, IX Encuentro REDBIO 2016-PERU, Lima, Peru 6/28/2016 19. "The potential of gene editing in animal agriculture", Presentation to a group of New Zealand pastoral producers, Palo Alto, CA 7/8/2016 20. "Gene editing: Breeding or Genetic Engineering?", Google hangout - 1 hr webinar 8/17/2016 21. "Gene editing: Breeding or GMO?", 11th International Marine Biotechnology Conference, Baltimore, MD 8/31/2016 What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we plan to continue to develop an efficient approach to performing a gene knock-in of SRY in bovine embryos. Once a gRNA is selected using the assay optimized for the MDBK cell line and verified through microinjection of the gRNA and Cas9 protein in bovine embryos, we will develop a donor vector containing the SRY coding sequence, the SRY and SP1 promoters and 1kb homology arms to begin performing the gene knock-in. Recent studies have shown that incubating the embryos in Scr7, an inhibitor of NHEJ and RS-1, an activator of homology directed repair (HDR), increases the efficiency of performing a gene knock-in using this system in mice and rabbits. We will test various concentrations of these two small molecules to determine which concentrations result in the highest knock-in efficiency, while yielding the highest success rate of embryo production. Once we have optimized this system, we will begin transferring XSRYY confirmed embryos into surrogate heifers. After the XSRYY bull calves are born, we can begin to evaluate the effects of copy number variation in SRY on fertility of XSRYY bulls. Additionally, once the XSRYY bulls reach sexual maturity, we will mate them to heifers and analyze the offspring to determine if this single gene knock-in of the endogenous bovine SRY gene (XSRY) is sufficient and necessary to produce all male (½ XY and ½ XXSRY) offspring. Finally, we will continue to video document the steps involved in this project in order to develop extension and public outreach materials that will provide information about gene editing technologies and animal biotechnology.

Impacts
What was accomplished under these goals? One of the primary concerns with the creation of genetically engineered (GE) organisms as a means to increase the food supply is the potential disruption of natural ecosystems. However, with advancements in gene editing, such as the discovery of the CRIPSR/Cas9 system, it is possible to develop a system for biological genetic containment through sterility in mammals, while also developing an approach to improve the efficiency of beef production. The overall goal of this project is to generate cattle with an SRY knock-in via CRISPR technology that will enable them to produce only male offspring, which are more desirable from a production standpoint, with the added feature of containing the transgene to a single generation since those male offspring will presumably be sterile. Objective 1. Develop an efficient gene knock-in approach using the CRISPR/Cas9 system to generate cisgenic bulls carrying an extra copy of the endogenous bovine SRY gene in the non-pseudoautosomal region of the X chromosome (XSRY) (Year 1) - 50% completion During this reporting period, the primary focus has been to develop and optimize an efficient gene knock-in approach using the clustered regularly interspersed short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system in a bovine cell line, as well as in bovine embryos. Prior to optimizing this approach, the graduate student working on this project first developed the skills necessary to perform work in cell culture, follicular aspiration of live animals and in vitro embryo production, along with microinjections in single cell embryos. Once these skills were developed, a system for testing guide- RNAs (gRNAs) using Lipofectamine and electroporation transfection systems in Madin-Darby bovine kidney (MDBK) cells was optimized. The CRISPR-Cas9 system uses a gRNA to target a specific region of the genome, which results in the Cas9 endonuclease cleaving the target region and causing a double stranded DNA (dsDNA) break. This dsDNA break is then repaired by the cell's DNA repair mechanism, non-homologous end joining (NHEJ), which sticks the two blunt ends of the break back together. This usually results in the insertion or deletion (indels) of bases due to the low fidelity of this process. Four gRNAs were developed using CRISPR RGEN tools out of the Center for Genome Engineering, Institute for Basic Science, Korea. DNA was extracted from the MDBK cells and the region of interest was amplified by polymerase chain reaction (PCR) using primers designed in Primer3 software. The Surveyor assay, which hybridizes PCR products from cells that were not transfected with the CRISPR-Cas9 system with PCR products from cells that were transfected with this system, was then used to test for the presence of indels. When the gRNA results in successfully guiding the Cas9 endonuclease to the target location, indels in the PCR product from the transfected cells result in the presence of a bulge when hybridized with PCR product from the non-transfected cells. This bulge can then be detected by the Surveyor endonuclease and cleaved. This cleaved product is visualized by agarose gel electrophoresis and the intensity of the cleaved PCR product can be measured to determine the cleavage efficiency of the Cas9 endonuclease with each specific guide. To date, this assay has detected two gRNAs that resulted in cleavage of the target region. However, the cleavage efficiency of the Cas9 endonuclease with these two guides is 33.3% and 21.7%, respectively. While this efficiency may be suitable for performing a gene knockout, this project will require a guide that results in higher cleavage efficiency in order to perform a knock-in of the SRY gene. In addition to testing guides using this assay, we have microinjected the gRNA with Cas9 protein in bovine embryos to determine the cleavage efficiency of each of these guides. To accomplish this, the embryos were allowed to continue to develop to the blastocyst stage after microinjection, at which point they were transferred into lysis buffer. The region of interest was amplified in the blastocyst DNA by PCR, purified, and Sanger-sequenced. This sequence was then compared to reference and control sequences in order to detect the presence of indels. Similar to the efficiencies of the gRNAs determined in the cell line, we have so far only detected cleavage efficiency in the 20-30% range, which is not ideal for performing a knock-in in bovine embryos. In order to improve cleavage efficiency, we are currently using the new bioinformatics tool out of the Church Lab at Harvard, sgRNA Scorer 1.0, to develop new guides. We anticipate that this updated tool will enable us to quickly select and test new guides. Objective 2. Evaluate the effects of copy number variation in SRY on fertility of XSRYY bulls (Year 2) - 0% completion Objective 3. Determine if this single gene knock-in of the endogenous bovine SRY gene (XSRY) is sufficient and necessary to produce all male (1⁄2 XY and 1⁄2 XXSRY) offspring from XSRYY bulls (Year 3) - 0% completion Objective 4. Develop extension and public outreach materials that will provide information about gene editing technologies and animal biotechnology (Year 3) - 10% completion While objective 4 will primarily be completed in year 3, we have video documented the methods and procedures involved in research surround the development of an efficient approach to performing a gene knock-in using bovine embryos. Based on our progress, we expect to develop an efficient method for performing a knock-in of SRY into the non-pseudoautosomal region of the X chromosome in bovine embryos and have the embryos transferred into surrogate heifers by the end of the year. This will allow us to evaluate the effects of copy number variation in SRY on fertility of XSRYY bulls, determine if this single gene knock-in of the endogenous bovine SRY gene (XSRY) is sufficient and necessary to produce all male (1⁄2 XY and 1⁄2 XXSRY) offspring from XSRYY bulls, and to develop extension and public outreach materials that will provide information about gene editing technologies and animal biotechnology within the timeframe outlined above.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Van Eenennaam, A.L. 2016. The Current and Future Uses of Biotechnology in Animal Agriculture Ceiba 54(1): 72-81. http://www.lamjol.info/index.php/CEIBA/article/view/2782/2531
  • Type: Other Status: Published Year Published: 2016 Citation: Genetic containment of livestock via CRISP-mediated gene knock-in. Poster presented at NIFA BRAG investigators meeting, June 2016