I n the modern US dairy cattle industry, destruction of hornproducing cells before they grow and attach to the skull (disbudding) is a routine practice to prevent horn growth. Animals that do not have horns do not injure other animals, require less feeding trough space, are less dangerous to handle and transport than horned animals and have fewer aggressive behaviors 1. Disbudding is an unpleasant process that has important implications for animal welfare, and many stakeholder groups have campaigned for alternative, humane solutions. One option is to select and breed animals that do not have horns, a phenotype referred to as polled. In 2016, Carlson et al. 2 reported the introgression of the P C Celtic POLLED allele into two male dairy bulls by genome editing using transcription activator-like effector nucleases (TALENs). Bulls RCI001 and RCI002 originated at Recombinetics, Inc., where the researchers genome-edited donor cells from a University of Minnesota crossbred dairy bull and then used reproductive cloning. Whole-genome sequencing (WGS) did not reveal any off-target alterations 2 , and both bulls reached maturity without developing horns. These genome-edited polled bulls were transferred to the University of California (UC), Davis and generated widespread interest. However, further work needs to be done in characterizing these animals if genome editing is to seamlessly integrate into livestock genetic improvement programs. Edits will likely need to be introduced into multiple elite founder animals to prevent genetic bottlenecks 3. Perhaps as importantly, appropriate regulatory frameworks that are risk-and evidencebased, proportionate and globally harmonized will be essential to allow research to occur, and to foster the development of useful applications 4. Others have reported on WGS of trios of genomeedited (CRISPR/Cas9) knockout livestock produced through cytoplasmic injection (CPI) of guide RNA (gRNA) and Cas9 into one-cell-stage zygotes. Genome-edited sheep were compared to their parents 5 and genome-edited goats were compared to their offspring 6 , and both trio-based studies concluded that de novo mutation rates were comparable to those observed in nonedited trios. A third study used an unbiased WGS on two genome-edited calves produced by a targeted gene knockout of beta-lactoglobulin using CPI of a homology-directed repair (HDR) donor plasmid and TALENs into early zygotes 7. These calves were free of any TALENmediated off-target mutations or donor plasmid integration events. To provide data to guide emerging regulatory frameworks and benefit future applications of genome editing in livestock, we set up a breeding experiment to investigate whether the POLLED genome edit was faithfully passed to offspring and whether there were any unique phenotypic or genotypic changes in those offspring. The calves produced as part of the current study are, to our knowledge, the first reported offspring of a genome-edited bull. These data will help inform regulatory agencies as they formulate processes to regulate g...

One of the ultimate goals of regenerative medicine is the generation of patient-specific organs from pluripotent stem cells (PSCs). Sheep are potential hosts for growing human organs through the technique of blastocyst complementation. We report here the creation of pancreatogenesis-disabled sheep by oocyte microinjection of CRISPR/Cas9 targeting PDX1, a critical gene for pancreas development. We compared the efficiency of target mutations after microinjecting the CRISPR/Cas9 system in metaphase II (MII) oocytes and zygote stage embryos. MII oocyte microinjection reduced lysis, improved blastocyst rate, increased the number of targeted bi-allelic mutations, and resulted in similar degree of mosaicism when compared to zygote microinjection. While the use of a single sgRNA was efficient at inducing mutated fetuses, the lack of complete gene inactivation resulted in animals with an intact pancreas. When using a dual sgRNA system, we achieved complete PDX1 disruption. This PDX1−/− fetus lacked a pancreas and provides the basis for the production of gene-edited sheep as a host for interspecies organ generation. In the future, combining gene editing with CRISPR/Cas9 and PSCs complementation could result in a powerful approach for human organ generation.

Genome editing using programmable nucleases has revolutionized biomedical research. CRISPR-Cas9 mediated zygote genome editing enables high efficient production of knockout animals suitable for studying development and relevant human diseases. Here we report efficient disabling pancreatogenesis in pig embryos via zygotic co-delivery of Cas9 mRNA and dual sgRNAs targeting the PDX1 gene, which when combined with chimeric-competent human pluriopotent stem cells may serve as a suitable platform for the xeno-generation of human tissues and organs in pigs.

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