Source: Recombinetics, Inc. submitted to
IMPROVEMENT OF DIARY ANIMAL WELL-BEING BY GENETIC DEHORNING
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1005738
Grant No.
2015-67015-23316
Project No.
MINW-2014-06202
Proposal No.
2014-06202
Multistate No.
(N/A)
Program Code
A1251
Project Start Date
Apr 15, 2015
Project End Date
Apr 14, 2019
Grant Year
2015
Project Director
Sonstegard, T.
Recipient Organization
Recombinetics, Inc.
2575 University Avenue West, Suite 100
Saint Paul,MN 55114
Performing Department
(N/A)
Non Technical Summary
The majority of dairy cattle in production today have been dehorned early in life using mechanical or chemical agents to protect against injury to other cattle or their animal handlers. Both the act of dehorning and the methods used open the industry to criticism and scrutiny by citizens and Non-governmental organizations concerned with animal rights and welfare. The optimal solution to the problem, which is recommended by governments around the world, is to breed with cattle that naturally lack horns, a trait called polled. While this breeding scheme could be easily implemented, the majority of polled cattle are suboptimal for dairy production and would represent a significant setback in dairy production that would take decades to recover. There is a simple, direct, rapid solution - gene-editing. Gene-editing is a technology that can seamlessly combine the desired traits of two unrelated animals without crossbreeding, thus preserving the present day production from dairy cattle while eliminating horns with genetic methods. In this study, we will evaluate gene-editing as a means for genetically dehorning cattle without crossbreeding in three steps. First we will introduce the polled genetics into dairy cattle by gene-editing and determine if it has the same effect as crossbreeding. Second, we will determine if there are any unintended effects on the animals; and third, we will evaluate the feasibility of deploying this technology in a variety of breeds to enable broad acceptance and rearing of genetically dehorned cattle.
Animal Health Component
34%
Research Effort Categories
Basic
34%
Applied
33%
Developmental
33%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3153410104034%
3043410104033%
3033410104033%
Knowledge Area
303 - Genetic Improvement of Animals; 304 - Animal Genome; 315 - Animal Welfare/Well-Being and Protection;

Subject Of Investigation
3410 - Dairy cattle, live animal;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
Goal 1: To evaluate the putative dominant genetic element responsible for the Polled phenotype in cattle.· Introgress the Pc Polled allele into fibroblasts from horned Holstein cattle.· Create cloned animals from these cells· Monitor horn development and growth characteristics in clones and their offspringGoal 2: To evaluate the influence of gene-editing and cloning on livestock genomes.· Develop fibroblast lines of L1 Dominette 01449 with the Pc allele· Produce cloned animals with and without the Pc allele from same-treated fibroblasts· Sequence the genome of the source material and clones to evaluate the frequency of off-target effects/mutations due to gene-editing, cell culture and cloningGoal 3: To ascertain the utility and efficiency of direct non-meiotic Polled introgression into cattle embryos.· Compare the frequency of TALEN or CRISPR/Cas9 introgression of the Pc allele into cattle embryos· Establish the optimal tradeoff between introgression frequency and embryo developmentGoal 4: To evaluate the feasibility and economics of introgression of the Polled trait into dairy or beef breeds by gene-editing versus standard breeding. · Model breeding systems to introgress Pc into other breeds to determine:* How many edited cattle would be required for each breed* What is the best method to produce the animals* Economically, how does gene-editing compare to breeding for introgression of polled in a variety of breeds
Project Methods
This project will used gene-editing via TALENs or CRISPR/Cas9 to introgress the Pc allele into the genomes of naïve cattle and will further use cloning to produce live animals for phenotypic and genotypic evaluation. Milestone 1: Cattle with introgressed Pc alleles will not develop horns. Phenotypic evaluations will include measurement of standard growth parameters, clinical chemistries, blood counts, semen evaluation and comparison to non-edited clones or historic data. Milestone 2: Outside of horn development, cattle with introgressed Pc alleles will be indistinguishable from breed cattle. Genotypic evaluations will use next generation sequencing to determine if gene-editing leads to significant off-target effects in cattle with novel introgression alleles. This will be accomplished by editing cells from L1 Dominette 01449, the Herford cow from which the bovine reference sequence was created from, and evaluation of novel mutations incurred during the cell culture, editing or cloning processes. Milestone 3: Gene-editing has negligible effects on the genome of edited cattle. Next, we will evaluate direct embryo microinjection as a means to introgress the Pc allele into naïve cattle genomes. Both TALENs and CRISPR/Cas9 will be evaluated and conditions defined for the optimum balance between introgression rate and embryo development. Milestone 4: Introgression by embryo microinjection will be a feasible option for rapid dissemination of polled genetics in cattle. Finally, given the efficiency and cost parameters of the methods above and input from cattle genetics companies and producers, we will evaluate the best practices for introgression of Polled into a variety of cattle breeds and the economics of each approach. Milestone 5: Develop a list of breeds that would suitable candidates for dehorning by gene-editing versus breeding.

Progress 04/15/15 to 04/14/19

Outputs
Target Audience:Scientists, farmers, public, and regulators of biotechnology applications. The polled cattle were featured, as in years past, in a variety of media sources listed in this report. Press "OZY" This Geneticist Is Creating Gene-Edited Animals For Our Plates. 4/29/18 https://www.ozy.com/rising-stars/this-geneticist-is-creating-gene-edited-animals-for-our-plates/85700 "Progressive Dairyman - "Use of gene editing to introduce the polled trait into elite germplasm. 6/4/18 https://www.progressivedairy.com/topics/a-i-breeding/use-of-gene-editing-to-introduce-the-polled-trait-into-elite-germplasm "Dairy Herd Management" 'Use of Gene Editing to Introduce the Polled Trait Into Elite Germplasm. 6/12/18 https://www.dairyherd.com/article/use-gene-editing-introduce-polled-trait-elite-germplasm "Canadian Jersey Breeder" The Uses of Biotechnology to Improve Animal Welfare. Sept 2018 https://jerseycanada.com/jersey-breeder-magazine/ "Seed World" Cows and Gene-Editing: Q&A with Alison Van Eenennaam. Sept 2018 https://seedworld.com/cows-gene-editing-qa-alison-van-eenennaam/ "Pacific Standard Magazine" Why the FDA's Plan to Regulate Gene Editing in Animals Has Some Scientists Worried. 11/1/18 https://psmag.com/news/why-the-fdas-plan-to-regulate-gene-editing-in-animals-has-some-scientists-worried "Successful Farming" 10 Predictions For The Future Of Gene Editing In Livestock. 11/29/18 https://www.agriculture.com/livestock/10-predictions-for-the-future-of-gene-editing-in-livestock Washington Post: Gene-edited farm animals are coming. Will we eat them?12/17/18 https://www.washingtonpost.com/news/national/wp/2018/12/17/feature/gene-edited-farm-animals-are-coming-will-we-eat-them Dairy Herd Management: Gene Editing finds its Way to the Farm12/21/18 https://www.dairyherd.com/article/gene-editing-finds-its-way-farm Flash Forward Podcast: Snip Snip Snip12/18/18 https://www.flashforwardpod.com/2018/12/18/snip-snip-snip/ Synthego Podcast: Alison Van Eenennaam Edits Cattle Using CRISPR12/4/18 https://www.synthego.com/blog/alison-van-eenennamm-podcast "The Conversation: Gene-edited food regulations: whether it's a plant or animal shouldn't matter, but it does now" 2/26/19 https://theconversation.com/gene-edited-food-regulations-whether-its-a-plant-or-animal-shouldnt-matter-but-it-does-now-112358 The Daily Beast: Is a Gene-Edited Animal a Drug? 2/27/19 https://www.thedailybeast.com/is-a-gene-edited-animal-a-drug Futurism.com: Scientists Are Fleeing America's Rules About Gene-Editing Animals3/15/19 https://futurism.com/scientists-fleeing-america-gene-editing-animals "Wired: A more human livestock industry, brought to you by CRISPR" 3/19/19 https://www.wired.com/story/crispr-gene-editing-humane-livestock/ Genetic Literacy Project Viewpoint: FDA plan to regulate gene-edited animals as drugs will thwart US food innovation 4/8/19 https://geneticliteracyproject.org/2019/04/08/viewpoint-fda-plan-to-regulate-gene-edited-animals-as-drugs-will-thwart-us-food-innovation/ Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Completed training of one Master's Student at UC-Davis. This person is now entered into the UCD PhD program. Other technical staff at Recombinetics and UC-Davis was trained in gene editing for polled at the celtic locus. How have the results been disseminated to communities of interest?There have been numerous invited scientific presentations and press articles. The 16 press article links for 2018 and 2019 are listed in a previous page. There were also presentations at stakeholder and scientific meetings, which included more than 70 presentations and11 abstracts respectively. All the sequencing information from this project is publicly available through the short read archive. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Update on two gene edited polled calves (April 15, 2018 - April 14, 2019) RCI 002 was euthanized and incinerated as an unapproved new animal drug 6/7/2018. His frozen semen, packaged for exportation and deployment, was approved for import into Brazil and Australia. Eleven calves were born in Australia in 2019; all of which were polled and derived from horned, purebred Holstein heifers. The introduction of the Polled trait into the U.S. dairy cattle population (Holstein and Jersey dairy breeds) by gene-editing versus standard breeding was simulated to evaluate the economic feasibility and published in the Journal of Dairy Science. Additionally, the introduction of the Polled trait into the Northern Australian beef cattle population (Brahman breed) by gene-editing versus standard breeding was simulated and results are currently being analyzed for publication. During this reporting period, a number of presentations, as well as abstracts, proceedings and many media pieces, discussed this technology and these particular animals.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Maci L Mueller, John B Cole, Tad S Sonstegard, Alison L Van Eenennaam, Simulation of introgression of the POLLED allele into the Jersey breed via conventional breeding vs. gene editing, Translational Animal Science, Volume 2, Issue suppl_1, September 2018, Pages S57S60, https://doi.org/10.1093/tas/txy054
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Mueller, M. L., J. B. Cole, T. S. Sonstegard, and A. L. Van Eenennaam. (2019). "Comparison of gene editing versus conventional breeding to introgress the POLLED allele into the US dairy cattle population." Journal of Dairy Science 102(5):4215-4226. https://doi.org/10.3168/jds.2018-15892 (May 2019 Editors Choice Paper for Production: Breeding, Genetics, and Genomics)
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: M.L. Mueller, J.B. Cole, N.K. Connors, D.J. Johnston, I.A.S. Randhawa, and A.L. Van Eenennaam. 2019. Comparison of gene editing versus conventional breeding to introgress the POLLED allele into the tropically-adapted Australian beef cattle population. Proc. Assoc. Advmt. Anim. Breed. Genet: 23: In press.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Mueller, M. L., and E. Yeiser Stepp. 2018. "Issue Brief: Polled Genetics." National Milk Producer's Federation (NMPF) - F.A.R.M Animal Care Program.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Mueller, M. L., J. B. Cole, T. S. Sonstegard, and A. L. Van Eenennaam. (2018). Comparison of gene editing vs. conventional breeding to introduce the POLLED allele into the U.S. dairy cattle population. Proc. Large Animal Genetic Engineering Summit, Park City, UT.
  • Type: Theses/Dissertations Status: Accepted Year Published: 2019 Citation: Mueller, M. L. 2019. Comparison of Gene Editing versus Conventional Breeding to Introgress the POLLED Allele into Dairy and Beef Populations. Unpublished master's thesis. University of California, Davis, CA.


Progress 04/15/17 to 04/14/18

Outputs
Target Audience:Scientists, farmers, public, and regulators of biotechnology applications. The polled cattle were featured, as in years past, in a variety of media sources listed in this report. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One Master's student was trained through this project; and that student obtained her Master's with an emphasis on animal biotechnology applications for genetic improvement in May of 2018. How have the results been disseminated to communities of interest?Presentations by Professor Alison Van Eenennaam "Impacts of the Revolutionary Technology (CRISPR & other biotech) on Crop and Livestock Agriculture and Bio-Engineering Value Chains", Strategic Perspectives on Innovation in Agrifood Supply Chains: Profitability, Sustainability, and Global Change, UC Berkeley, Berkeley, CA 4/18/17 "Regulatory environment for new technologies", The Analytical Excellence Through Industry Collaboration (AEIC) Spring Meeting 2017, Santa Clara, CA 4/19/17 "The #Scicomm challenge facing animal agriculture", New Horizons Seminar Series, School of Veterinary Medicine, Davis, CA 4/26/17 "Gene Editing in Animal Breeding", GeneSeek Science Advisory Council 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 Improvement of Food Animals: Past and Future", International Consortium on Applied Bioeconomy Research (ICABR) 5/31/2017 "Advancements in emerging technology: How genome editing could synergistically accelerate animal genomics", Beef Improvement Federation Conference, Athens, GA 6/2/2017 "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 Biotechnolog", International Society of Animal Genetics, 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 "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 "The Future of Genetic Alteration in Food Animal Production", 50th Annual Conference of the American Association of Bovine Practitioners, Omaha, NE 9/15/2017 "Gene Editing: The Polled Phenotype in Dairy Cattle as a Test case for Social License", Breeding for Efficiency 2017 EDGP Symposium, Guelph, Ontario, Canada 10/16/2017 "Proposed Regulation of Gene Edited Animals in the U.S.", Netherlands Commission on Genetic Modification (COGEM), Rotterdam, The Netherlands 10/20/2017 "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 "Animal Biotechnology: A Tool for Good or Evil?" UC Davis Scinema, Davis, CA 11/7/2017 "Regulatory oversight of new breeding innovations in the U.S.", TropAgBio2017, Queensland, Australia 11/19/2017 "Science communication to obtain social license for use of genome editing in animal breeding programs", The Candian Agriculture and Agri-Food Genomics Forum, Toronto, Canada 11/30/20 "Innovation in Agricultural Science", 2017 Agribusiness Roundtable, Tempe, AZ 12/4/2017 "Innovation in Agricultural Science?" 2018 Legislative Agriculture Chairs Summit, State Agriculture and Rural Leaders & Council of State Governments, Kansas City, MO 1/6/2018 "Gene Editing" American Farm Bureau Convention, Nashville, TN 1/8/2018 "Biotechnology - promise and politics" College of Life Sciences, Brigham Young University, Provo, UT 1/18/2018 "The Landscape of Gene Editing in Food and Agriculture", Center for Food Integrity (CFI) Gene Editing Summit, Arlington, VA 3/6/2018 "Use of gene editing to introduce the polled trait into elite germplasm", Golden State Dairy Conference, Stockton, CA 3/29/2018 "Genome editing applications in animals", Genome Editing Seminar for Biotechnology Regulators in the Americas, Center for Tropical Agriculture, Cali, Columbia 4/4/2018 Abstracts Van Eenennaam, A.L. 2016. "The future of genetic alteration in food animal production", American Association of Bovine Practitioners, Omaha, NE 9/14-16/2017 Van Eenennaam, A.L. 2016. "Proposed Regulation of Gene Edited Animals in the U.S.", Netherlands Commission on Genetic Modification, Rotterdam, The Netherlands 10/20/2017 Van Eenennaam, A.L. "Regulatory oversight of new breeding innovations in the U.S.", TropAg2017, Brisbane, Australia 11/20-22/2017 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 Mueller, M.L., Cole, J.B., Sonstegard, T.S., Van Eenennaam, A.L. "Simulation of Introgression of the Polled Allele into the Holstein Breed via Conventional Breeding versus Gene Editing", 11th World Congress on Genetics Applied to Livestock Production, Auckland, New Zealand 2/11-16/2017 Press Digital Trends, April 17, 2017, http://www.digitaltrends.com/cool-tech/crispr-gene-editing-and-the-dna-of-future-food/#ixzz4eYcMJK7C interestingthingsonline.com, April 18, 2017, http://www.interestingthingsonline.com/from-corn-to-cattle-gene-editing-is-about-to-supercharge-agriculture/ AgFront, June 2017, http://www.agfront.com/2017/06/16/genome-editing-whats-big-deal/#.WiBKAkqnGUk The Land, July 10, 2017, https://www.theland.com.au/story/4779762/how-gene-editing-could-change-livestock/ The Athens Science Observer, July 22, 2017, https://athensscienceobserver.com/2017/07/22/seeking-greener-pastures-gene-editing-and-animal-welfare/ Cameron J. English podcast, September 10, 2017, https://www.cameronjenglish.net/single-post/2017/09/11/Episode-16-Solving-the-worlds-problems-with-genetics-Dr-Alison-Van-Eenennaam Bloomberg Businessweek, October 12, 2017, https://www.bloomberg.com/news/articles/2017-10-12/this-genetics-company-is-editing-horns-off-milk-cows Science Friction, ABC Radio National, Australian Broadcasting Corporation, November 18, 2017, http://www.abc.net.au/radionational/programs/sciencefriction/making-happier-animals-gene-editing-on-the-farmyard/9154208 Missouri Farm Bureau, January 1, 2018, https://www.mofb.org/NewsMedia/Commentary.aspx?articleID=803&articleYear=2018 Tri-State Livestock News, January 11, 2018, https://www.tsln.com/news/cattle-health/the-present-and-future-of-cattle-breeding-dna-testing-ge-epds-gene-editing/ Oklahoma Farm Report, January 15, 2018, http://www.oklahomafarmreport.com/wire/news/2018/01/00987_BeefBuzzVanEenanaamGeneEditingMisconceptions01152018_134223.php#.Wx79YEgvy70 AgriPulse, January 29, 2018, https://www.agri-pulse.com/articles/10527-protecting-the-herd-new-opportunities-through-gene-editing The Fish Site, February 6, 2018, https://thefishsite.com/articles/benchmark-gene-editing-interview GenomeWeb, February 9, 2018, https://www.genomeweb.com/gene-silencinggene-editing/genome-editing-reaching-farm-animals-though-regulatory-uncertainty-looms#.Wx76nUgvy70 MIT Technology Review, March 12, 2018, https://www.technologyreview.com/s/610027/farmland-gene-editors-want-cows-without-horns-pigs-without-tails-and-business-without/ The Warren Centre, March 16, 2018, https://thewarrencentre.org.au/no-horns-no-tails-and-no-regulations/ WND, March 22, 2018, http://mobile.wnd.com/2018/03/new-push-to-deregulate-gene-edited-animals/ Innovation Files, Information Technology & Innovation Foundation, April 3, 2018, https://itif.org/publications/2018/04/03/gene-editing-government-regulation-and-greening-our-future What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The Dominette polled cell ine was developed; and these cells were sent to the University of Adelaide for cloning in 2018. New Cas9/CRISPR combinations for making double strand breaks at the site for Pc introgression were designed and tested in fibroblast culture and on in vitro produced embryos. The purpose was to determine if higher effciencies of cutting could be obtained compared to TALENs, and higher eficiencies (>10%) were obtained. As previously reported, RCI 003 was processed at the UC Davis Department of Animal Science Meat Lab in October, 2016 under standard procedures. Tissue samples were collected and stored and the carcass and meat were sent to Recombinetics, Inc. Eurofins Laboratory testing of the meat found it equivalent in compsition and matabolites to that from Angus grade by USDA as select. Semen forr production of artificial inseminationstraws was collected from RCI 002. The introduction of the Polled trait into the U.S. dairy cattle population (Holstein and Jersey dairy breeds) by gene-editing versus standard breeding was simulated to evaluate the economic feasibility; thus completing Goal 4. During this reporting period, a number of presentations, as well as abstracts, proceedings and many media pieces, discussed this technology and these particular animals. A full list of presentations and publications can be found at the end of this report.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Van Eenennaam, A.L. 2018. The Importance of a Novel Product Risk-Based Trigger for Gene-Editing Regulation in Food Animal Species. The CRISPR Journal 1(2): 1-6.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Mueller, M.L., Cole, J.B., Sonstegard, T.S., Van Eenennaam, A.L. Simulation of Introgression of the Polled Allele into the Holstein Breed via Conventional Breeding versus Gene Editing, 11th World Congress on Genetics Applied to Livestock Production, Auckland, New Zealand 2/11-16/2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Van Eenennaam, A.L. 2016. The future of genetic alteration in food animal production, American Association of Bovine Practitioners, Omaha, NE 9/14-16/2017


Progress 04/15/16 to 04/14/17

Outputs
Target Audience:Scientists, farmers, public ect. The polled cattle were fetured in a variety of media sources, listed below. Changes/Problems:We were previously held up by insufficient semen production, a problem that was overcome in the last year. Secondly, the Dominette cells were difficult to work with and led to delays in production. We had temporarily switched to Domino cell lines prior to developming new techniques that were effective in Dominette cells. Though this led to a delay in the proposed timeline, the issues have been overcome and funds remain in the budget to complete the work. What opportunities for training and professional development has the project provided?Training of 1 Ph.D. student in advanced reproductive techniques and preparation of editing tools for microinjection. Also, continued training of a Ph.D. student for bioinformatic analysis of whole genome resequencing data for finding off-site or unintended edits from live animal genomes. One Master's student has developed microinjection skills and how to assess editing in blastocysts staged embryos. How have the results been disseminated to communities of interest?An initial paper describing the polled phenotype of Spotigy and Buri (cloned prior to grant submission) was published in Nature Biotechnology. "Production of hornless dairy cattle from genome-edited cell lines." Nat Biotechnol. 2016 May 6;34(5):479-81. doi: 10.1038/nbt.3560. One poster presentation at the USDA Annual meeting in Chicago IL. Numerous mentions at invited talks (itemized below) and popular press articles. Presentations Van Eenennaam, A.L. "The Current and Future Uses of Biotechnology in Animal Agriculture", Ensminger University Conference, Honduras, 5/14/16 Van Eenennaam, A.L. "Emerging Genetic Advancements", Center for Food Integrity Conference, Hamburger University, McDonald's Campus, Chicago, IL, 5/18/16 Van Eenennaam, A.L. "Animal Biotechnology", BIO 2016 FutureMakers Ted-style talk, BIO convention, San Francisco, CA 6/8/16 Van Eenennaam, A.L. "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 Van Eenennaam, A.L. "The potential of gene editing in animal agriculture" Presentation to group of New Zealand pastoral producers. Palo Alto, CA 7/8/2016 Van Eenennaam, A.L. "Gene editing: Breeding or Genetic engineering?" Riverview LLP Cattle Company, Google Hangout, 8/17/2016 Van Eenennaam, A.L. "Gene editing: Breeding or GMO?" 11th International Marine Biotechnology Conference (IMBC), Baltimore, MD, 8/31/2016 Van Eenennaam, A.L. "Gene editing: Breeding or GMO?" Genome Editing and the Future of Farming, Edinburgh, Scotland 9/6/2016 Van Eenennaam, A.L. "Animal Biotechnology: Opportunities and Challenges" Cornell Alliance for Science Fellows Training Program, Ithaca, NY 9/13/2016 Van Eenennaam, A.L. "Gene editing: what is it and how might it be used in animal breeding programs" American Wagyu Association Conference, Coeur d'Alene, ID 9/15/2016 Van Eenennaam, A.L. "Gene editing: Breeding or Genetic engineering?" Biotech University, Saskatoon, Saskatchewan, Canada, 9/29/2016 Van Eenennaam, A.L. "Using genome editing to introgress desirable intragenic and intergenic alleles to accelerate the rate of gain in meat animal breeding programs" XVI Latin American Genetics Congress, Montevideo, Uruguay, 10/10/2016 Van Eenennaam, A.L. "Genome editing: What's all the fuss?" NBCEC Brown Bagger Series, webinar, 10/12/2016 Van Eenennaam, A.L. "Genome editing: What's all the fuss?" NBCEC Brown Bagger Series, webinar, 10/12/2016 Van Eenennaam, A.L. "Genome Editing" National Association of Animal Breeders (NAAB) 70th Annual Convention, Green Bay, WI, 10/14/2016 Van Eenennaam, A.L. "Animal Biotechnology: What is, what could be, and will it be?" Marian Koshland Memorial Lecture, Berkeley, CA, 11/29/2016 Van Eenennaam, A.L. "Genomics 101 and potential uses of gene editing in the California Beef Industry" IN-service training on genetic evaluation in beef cattle, UCCE beef cattle workgroup meeting at California Cattlemen's Associaiton meeting, Reno, NV, 12/1/2016 Van Eenennaam, A.L. "Do Kingdoms Matter? Regulation of Animal Breeding Technologies" International Plant and Animal Genome Conference, San Diego, CA, 1/15/2017 Van Eenennaam, A.L. "Gene Editing - The Pros and Cons and Relevance to the Beef Sector" British Cattle Breeder's Club, Telford, Shropshire, England, 1/24/2017 Van Eenennaam, A.L. "Animal Biotechnology: What is, what could be, and will it be allowed?" York Distinguished Lecture, Auburn University, Auburn, AL, 2/14/2017 Van Eenennaam, A.L. "How Genome Editing Can Synergistically Accelerate Animal Genetic Improvement Programs" AAAS 2017 Annual Meeting, Boston, MA, 2/17/2017 Van Eenennaam, A.L. "Will gene editing face the same fate as genetic engineering? The #scicomm challenge" AgriBio, LaTrobe University, Bundoora, Victoria, Australia, 3/21/2017 Abstracts Van Eenennaam, A.L. 2016. "Gene Editing: Breeding or GMO?" 11th International Marine Biotechnology Conference (IMBC), Baltimore, MD, 8/31/2016 Van Eenennaam, A.L. 2016. "Using genome editing to introgress desirable intragenic and intergenic alleles to accelerate the rate of genetic gain in meat animal breeding programs" XVI Latin American Genetics Congress, Montevideo, Uruguay, 10/10/2016 Van Eenennaam, A.L. "Do Kingdoms Matter? Regulation of Animal Breeding Technologies" International Plant and Animal Genome Conference, San Diego, CA, 1/15/2017 Van Eenennaam, A.L. "How Genome Editing Can Synergistically Accelerate Animal Genetic Improvement Programs" AAAS 2017 Annual Meeting, Boston, MA, 2/17/2017 Proceedings Van Eenennaam, A.L. 2016. "The Current and Future Uses of Biotechnology in Animal Agriculture" Ceiba, 54(1): 72-81. Van Eenennaam, A.L. "Gene editing: Breeding or GMO?" Genome Editing and the Future of Farming, Edinburgh, Scotland 9/6/2016 Van Eenennaam, A.L. 2016. "Using genome editing to introgress desirable intragenic and intergenic alleles to accelerate the rate of genetic gain in meat animal breeding programs" XVI Latin American Genetics Congress, Montevideo, Uruguay, 10/10/2016 Van Eenennaam, A.L. "Genome Editing" National Association of Animal Breeders (NAAB) 70th Annual Convention, Green Bay, WI, 10/14/2016 Van Eenennaam, A.L. "Gene Editing - The Pros and Cons and Relevance to the Beef Sector" British Cattle Breeder's Club, Telford, Shropshire, England, 1/24/2017 Journal articles 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 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 Van Eenennaam, A.L., 2017. Genetic Modification of Food Animals. Current Opinion in Biotechnology 44: 27-34, http://www.sciencedirect.com/science/article/pii/S0958166916302348 Press >75 publications- full bibliograpy exceeds space limits. "Polled (Hornless) - occurs naturally but now can be achieved by GM (Genetic Modification, Dairy Crossbreeding, May 2, 2016, http://www.dairycrossbreeding.com/polled-hornless-gaining-popularity-and-occurring-naturally/ "Regulate genome-edited products, not genome editing itself", Carroll et al., Nature, May 6, 2016, http://www.nature.com/nbt/journal/v34/n5/full/nbt.3566.html "Cows with horns may soon be a relic of farming's painful past", CapitalDom, May 6, 2016, http://www.capitaldom.com/cows-with-horns-may-soon-be-a-relic-of-farmings-painful-past "Gene-edited cattle produce no horns", Science magazine, Ben Panko, May 10, 2016, http://www.sciencemag.org/news/2016/05/gene-edited-cattle-produce-no-horns "Genetically Engineered Hornless Dairy Calves", Jef Akst, The Scientist, May 10, 2016, http://www.the-scientist.com/?articles.view/articleNo/46064/title/Genetically-Engineered-Hornless-Dairy-Calves/ "Gene Editing Makes Cows Without Horns With No Apparent Negative Effects", Charles Q. Choi, Popular Science, May 11, 2016, http://www.popsci.com/gene-editing-makes-cows-without-horns?dom=rss-default&src=syn Cows with horns may soon be a relic of farming's painful past", Kevin Loria, Tech Insider, May 12, 2016, http://www.techinsider.io/recombinetics-genetically-edited-cattle-without-horns-2016-5 "Gene editing to create hornless cattle", Farming Today, BBC Radio 4, Jun 6, 2016, http://www.bbc.co.uk/programmes/b07dlww0 Scientists Develop a Hornless Cow Through Gene Editing", NPR Science Friday, October 14, 2016, http://www.sciencefriday.com/segments/scientists-develop-a-hornless-cow-through-gene-editing/ What do you plan to do during the next reporting period to accomplish the goals?We will initiate production of the polled Dominette clones and also begin to test the HDR templates along with TALENs and CRISPRs using IVF methods. Whole genome resequencing was done on the first calves born, and we did show no detection of off-site targeting using TALENs. This will be repeated once the Dominette clones are born. All other goals are on schedule.

Impacts
What was accomplished under these goals? Under Goal 1 - All the bulleted items were completed 100%. The impact of the first cloned and genetically dehorned cattle being born and evaluated was ground-breaking, as evidenced by the enormous amount of popular press coverage and invitations to speak. *note- the above is from the previous report (2016). The last portion of bullet 3 mentions evaluation of offspring. Breeding from Buri has been successful and 6 calves are expected in September of 2017. Blastocysts from the same breeding were proven to carry the Pc allele. Hence, this milestone is 50% complete and will be completed in 2017. Under Goal 2 - Bulleted item one was previously stalled due to biological difficulties with Dominette cells. We have altered templates and since bene able to introgress Pc into Dominette. These cells, are scheduled up for cloning. The final goal cannot be completed until the clones are born. Under Goal 3 - The milestones are 50% and 30% completed. The TALEN introgression studies have demonstrated cutting without an HDR template are now are being conducted with a template. CRISPR injections have proven effective for cutting the allele and will be tested with HDR templates shortly. This work will be finalized by the end of 2017. Under Goal 4 - The milestone is 30% completed with no accomplishment to report. This milestone and goal was initially scheduled for the last year of the grant and are ramping up for completion by the end of the performance period.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Production of hornless dairy cattle from genome-edited cell lines. Nat Biotechnol. 2016 May 6;34(5):479-81. doi: 10.1038/nbt.3560.


Progress 04/15/15 to 04/14/16

Outputs
Target Audience:The following is a list of articles published about our work either with our input or reported by othersfor the general public and trade journals. Presentations Van Eenennaam, A.L. "Gene Editing: Breeding or Genetic Engineering?" Plant and Animal Genome XXIV. San Diego, CA 1/09/2016 Fahrenkrug, S.C. "Accelerated Breeding with Non-meiotic Allele Introression"Plant and Animal Genome XXIV. San Diego, CA 1/09/2016 Abstracts Van Eenennaam, A.L. 2016. "Gene Editing: Breeding or Genetic Engineering?" [abstract]. Plant and Animal Genome Conference XXIV, Jan 9-13, San Diego, CA. https://pag.confex.com/pag/xxiv/meetingapp.cgi/Paper/19390 Photographic exhibition "Bulls without horns" by Aleksandra Domanovic displayed in Germany http://www.tanyaleighton.com/index.php?pageId=658&l=en You Tube video Precision Breeding Offers New Alternative to Dehorning Cattle https://www.youtube.com/watch?v=-Qks_LMmodw Press associated with the gene-edited polled calves April 15, 2015 - April 14, 2016 "Engineering Hornless Cows", Talking Biotech Podcast, June 14, 2015, http://www.talkingbiotechpodcast.com/?p=36 "Genetically Modified Animals Will Be On Your Plate In No Time", Sophia Chen, Wired, July 6, 2015, http://www.wired.com/2015/07/eating-genetically-modified-animals/ "Why animal rights activists should love genetically edited animals", Kevin Loria, Tech Insider, July 28, 2015, http://www.techinsider.io/why-animal-rights-activists-should-love-genetically-edited-animals-2015-7 "Disturbing animal cruelty: Torment of dehorning calves prompts gene edit rescue", Roz Zurko, Examiner.com, July 28, 2015, http://www.examiner.com/article/disturbing-animal-cruelty-torment-of-dehorning-calves-prompts-gene-edit-rescue "Hornless cattle will herald a gene-editing revolution on farms", Michael Le Page, New Scientist, August 12, 2015, https://www.newscientist.com/article/mg22730344-000-hornless-cattle-will-herald-a-gene-editing-revolution-on-farms/ "Scientists could end animal cruelty - unless the FDA and anti-GMO activists stop them", Daniel Bier, CapX, August 25, 2015, http://capx.co/scientists-could-end-animal-cruelty-unless-the-fda-and-anti-gmo-activists-stop-them/ "This Scientist Might End Animal Cruelty - Unless GMO Hardliners Stop Him", Kat McGowan, Mother Jones, September/October 2015, http://www.motherjones.com/environment/2015/07/fahrenkrug-genetic-modification-gmo-animals "Pet micro pigs? Chinese biotech firm says it will sell very small swine", Los Angeles Times, October 5, 2015, http://www.latimes.com/world/asia/la-fg-china-micropigs-20151005-story.html "Born (horn) free: Minnesota lab stops them before they grow", Amanda Proscia, Great Lakes Echo, October 15, 2015, http://greatlakesecho.org/2015/10/15/minnesota-lab-stops-horns-before-they-grow/ "Genetic engineers trying to create hornless cows", The Rural Blog, October 19, 2015, http://irjci.blogspot.com/2015/10/genetic-engineers-trying-to-create.html "Open Season Is Seen in Gene Editing of Animals", Amy Harmon, The New York Times, November 26, 2015, http://www.nytimes.com/2015/11/27/us/2015-11-27-us-animal-gene-editing.html?_r=0 "Salmon and Calves Reframe Biotech Debate", Joan Conrow, Cornell Alliance for Science, November 26, 2015, http://allianceforscience.cornell.edu/blog/salmon-and-calves-reframe-biotech-debate "Scientists and ethicists debate 'gene editing' for animal health, Arlene Weintraub, Fierce Pharma, December 4, 2015, http://www.fiercepharma.com/r-d/scientists-and-ethicists-debate-gene-editing-for-animal-health "NAS panel tackles - and is tackled by - genome editing in animals", Science magazine, December 18, 2015, http://www.sciencemag.org/news/2015/12/nas-panel-tackles-and-tackled-genome-editing-animals "First genetically edited cows arrive at UC Davis", The Sacramento Bee, December 20, 2015, http://www.sacbee.com/news/local/health-and-medicine/article50822850.html "Scientists using genetic editing to delete dairy cow horns", ANR News Blog, December 21, 2015, http://ucanr.edu/blogs/anrnews/index.cfm?tagname=Alison%20van%20Eenennaam "What is gene editing?" Alison Van Eenennaam, eBEEF fact sheet, Dec 23, 2015, http://articles.extension.org/pages/73389/what-is-gene-editing "UC-Davis studying gene editing in cattle", John Maday, Drover's, December 23, 2015, http://www.cattlenetwork.com/news/industry/uc-davis-studying-gene-editing-cattle "Cut and Paste Animals", Western Livestock Journal, January 1, 2016, http://npaper-wehaa.com/western-livestock-journal/2016/01/15/?g=print#?article=2686019 "CDQAP Ruminations: Dehorning and Economics", Michael Payne, January 6, 2016, http://cdrf.org/2016/01/06/5046/ "What Genetically Engineered Animal Will Land on Your Dinner Plate Next?", Adele Peters, Co.Exist, Jan 14, 2016, http://www.fastcoexist.com/3054655/what-genetically-engineered-animal-will-land-on-your-dinner-plate-next "UC Davis welcomes campus' first gene-edited calves, Emma Sadlowski, The Aggie, January 19, 2016, https://theaggie.org/2016/01/19/uc-davis-welcomes-campus-first-gene-edited-calves/ "CRISPR is coming to agriculture - with big implications for food, farmers, consumers and nature", Maywa Montenegro, Ensia, January 28, 2016, http://ensia.com/voices/crispr-is-coming-to-agriculture-with-big-implications-for-food-farmers-consumers-and-nature/ "Gene Editing in Animals a Reality", Physicians Plasma Alliance, February 2, 2016, http://www.physiciansplasma.com/gene-editing-in-animals-a-reality/ "'Edited' food: Is this obscure tool a game changer for GMOs?", Maywa Montenegro, GreenBiz, February 5, 2016, https://www.greenbiz.com/article/edited-food-obscure-tool-game-changer-gmos "Genetically engineered animals could be coming to a plate near you", Kristofor Husted, the Fence Post, February 16, 2016, http://www.thefencepost.com/news/20571125-113/genetically-engineered-animals-could-be-coming-to-a "In the Genes: Hereford breed investigates possibilities of precision breeding", Amanda Radke, February 18, 2016, http://www.tsln.com/news/20709337-113/asdasda "Genetic Engineering Makes a Big Advance, Prolife.ie, February 29, 2016, http://www.prolife.ie/personal-update-issue-description/genetic-engineering-makes-big-advance "Designer Genes", Reynold Eergen, Canadian Cattlemen, February 2016, https://issuu.com/fbcpublishing/docs/160129230519-201ba65e39b7440286cbb713b0835690/19 "Scientists Breed Pigs Resistant to a Devastating Infection Using CRISPR", Monique Brouillette, Scientific American, March 1, 2016, http://www.scientificamerican.com/article/scientists-breed-pigs-resistant-to-a-devastating-infection-using-crispr/ "Genetic editing and cattle", Reynold Bergen, Canadian Catttlemen: The Beef Magazine, March 1, 2016, http://www.canadiancattlemen.ca/2016/03/01/designer-genes/ "Editing Animals: Gene editing applications expanding with advancing technology", Greg Cima, JAVMA News, March 15, 2016, https://www.avma.org/News/JAVMANews/Pages/160315a.aspx "Genmanipulierte Nahrung", Mediathek, March 3, 2016, http://mediathek.daserste.de/Plusminus/Genmanipulierte-Nahrung/Das-Erste/Video?documentId=34015334&topRessort&bcastId=432744 "Welcome to the CRISPR zoo", Sara Reardon, Nature, March 9, 2016, http://www.nature.com/news/welcome-to-the-crispr-zoo-1.19537 "Gene editing applications expanding with advancing technology", Greg Cima, JAVMA news, March 15, 2016, https://www.avma.org/news/javmanews/pages/160315a.aspx "Genetic Editing Emerges", Wyatt Bechtel, Beef Today, Early Spring 2015, http://digitaledition.qwinc.com/article/Genetic+Editing+Emerges/1969197/251874/article.html "nano spezial: Zuchters Traum", 3sat Mediathek, April 7, 2016, http://www.3sat.de/mediathek/?mode=play&obj=57910 "How genetic editing will impact your dairy future", Kirk Sattazahn, Hoard's Dairyman, April 10, 2016, http://www.hoards.com/T16apr10-genetic-editing Changes/Problems:The two challenges have been the lack of semen production from the existing clones to show normal transmission of the alleles introgressed by editing and the poor quality of fibroblasts remaining from Dominette. This later challenge was overcome by replacing her cells with those of her sire. We hope to rollover the funds for cloning Dominette into the next fiscal year for use in cloning Domino. The gene transmission testing may have to be delayed until the end of year 3 into 2019 using the clone of Domino as the test sire. What opportunities for training and professional development has the project provided?Training of 1 Ph.D. studentin advanced reproductive techniques and prepration of editing tools for microinjection. Also, training of a Ph.D. student for bioinformatic analysis of whole genome resequencing data for finding off-site or unintended edits from live animal genomes. One Master's student was being trained in evaluation of the edited animal performance.No undergraduates are trained to date. How have the results been disseminated to communities of interest?A paper on the production of the first genetically dehorned animals is being prepared for submission to Nature Biotechnology. Two presentations at the PAG cattle workshop were given about the birth of these animals. What do you plan to do during the next reporting period to accomplish the goals?We will initiate production of the polled Domino clones and also begin to test the different editing platforms for deployment using IVF methods. Whole genome resequencing was done on the first calves born, and we did show no detection of off-site targeting using Talens. This will be repeated once the Domino clone is born. All other goals are on schedule.

Impacts
What was accomplished under these goals? Under Goal 1 - All the bulleted items were completed 100%. The impact of the first cloned and genetically dehorned cattle being born and evaluated was ground-breaking, as evidenced by the enormous amount of popular press coverage and invitations to speak. Under Goal 2 - Bulleted item one cannot be completed due to biological problems with existing cells. Cells of DOminette's sire were established to compelte this accomplishment. The remaining milestones for this goal are 10% and 0% completed. We expect these items will be completed at the end of 2017. Under Goal 3 - The milestones are 20% and 0% completed with no accomplishments to report. This work will be finalized by the end of 2017. Under Goal 4 - The milestone is 30% completed with no accomplishment to report. This milestone and goal could be completed by April 2017.

Publications