Precision Genome Engineering in Livestock Directed Genetic Modification Technologies: An Opportunity for Science-Based Regulatory Reform A Roundtable Workshop January 14, 2015

Precision Genetic Editing is a “Game Changer” High gain, low risk Engineering Genetic Engineering pre-2010 DNA sequences inserted randomly Genome Editing post-2010 Precision DNA changes Rate of HDR <10-7 Rate of HDR 0.4 4x106 X Precision genetics => 1bp change in 3 x 109 bp! Error rates (June 2014) are below minimal detection (10-4)

Better Nutrition & Improved Animal Welfare with Changing Environments Applications of Gene-Edited Animals Global Improved Breeds for local environments Better Nutrition & Improved Animal Welfare with Greater Diversity for Changing Environments Animal Models for Biomedical Testing Patient-Specific Tissues & Cells Personal

Livestock Gene Conversion with TALENs

TALEN-mediated SNP changes about 10% efficient (pigs) TALEN-mediated HDR for gene editing 45% efficient, 32% biallelic (pigs) TALEN-mediated SNP changes about 10% efficient (pigs) Cas9/CRISPR-mediated SNP gene-editing average <10% efficient (pigs) Tan et al (2013) PNAS

Gene-editing Technology Relative Risks Nature Biotech. (2013) 31: 794-802 Gene-editing Technology regulation uncertain Regulated Unregulated Low --- Likelihood of Unintended Change  High 6

Programmable endonucleases change the paradigm Used to introduce targeted mutations - Not GE Used to target gene insertion - GE Before regulating targeted mutagenesis consider this: Base pair difference between any two humans ranges from 6 X 106 to 24 X 106 (Jorde et al., (2004). Nature Genetics 36 (11s): S28–33; Tishkoff et al., (2004). Nature Genetics 36 (11s): S21–7) Will not be traceable, i.e. not different from natural mutation From Jim Murray, UC-Davis

Transgenic Livestock Development Lysostaphin, Lysozyme, Lactoferrin Jim Murray & Elizabeth Maga, UC – Davis Jim Murray & Elizabeth Maga, UC-Davis Mark Westhusin & Chuck Long, TAMU Matt Wheeler, Illinois Kevin Wells, University of Missouri Scott Fahrenkrug & Dan Carlson, Recombinetics Chris Rogers, Exemplar Avian Influenza Resistance Helen Sang, Roslin Aquadvantage Salmon, Aquabounty

Gene Edited Livestock WT Pitx3 Knockout No Human Stem Cells RELA African Swine Fever Virus Resistance Warthog to S. scrofus using TALEN in embryos (Roslin Institute & Recombinetics) Recombinetics: PCSK9 Gain-of-Function mutation using transgenic KO LDLR mutation introduced using TALEN in cell cultures Myostatin allele from Belgian Blue to Nelore (B. Taurus to B. Indicus); direct embryo modification using TALEN (TAMU & Recombinetics) Recombinetics WT Pitx3 Knockout No Human Stem Cells + Human Stem Cells

FDA Approved Transgenic Livestock Glofish ® 2003 Atryn® Recombinant Antithrombin, 2009

Regulation of GE Animals United States: Outcome based (in theory); (in truth) process based By FDA as a “New Animal Drug” Guidance-based only, i.e. no statutes Canada: Novel Foods Argentina, Australia, New Zealand: Process-based Europe: fiat Brazil: ???