Source: Recombinetics, Inc. submitted to
IMPROVEMENT OF DIARY ANIMAL WELL-BEING BY GENETIC DEHORNING
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
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, 2018
Grant Year
2015
Project Director
Fahrenkrug, S. C.
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
33%
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%
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 04/15/16 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