Cas9-expressing chickens and pigs as resources for genome editing in livestock

Genetically modified animals continue to provide important insights into the molecular basis of health and disease. Research has focused mostly on genetically modified mice, although other species like pigs resemble the human physiology more closely. In addition, cross-species comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR-Cas9. Here, we report the generation of transgenic chickens and pigs that constitutively express Cas9 in all organs. These animals are healthy and fertile. Functionality of Cas9 was confirmed in both species for a number of different target genes, for a variety of cell types and in vivo by targeted gene disruption in lymphocytes and the developing brain, and by precise excision of a 12.7-kb DNA fragment in the heart. The Cas9 transgenic animals will provide a powerful resource for in vivo genome editing for both agricultural and translational biomedical research, and will facilitate reverse genetics as well as cross-species comparisons

Acquiring Resistance Against a Retroviral Infection via CRISPR/Cas9 Targeted Genome Editing in a Commercial Chicken Line

Genome editing technology provides new possibilities for animal breeding and aid in understanding host-pathogen interactions. In poultry, retroviruses display one of the most difficult pathogens to control by conventional strategies such as vaccinations. Avian leukosis virus subgroup J (ALV-J) is an oncogenic, immunosuppressive retrovirus that causes myeloid leukosis and other tumors in chickens. Severe economic losses caused by ALV-J remain an unsolved problem in many parts of the world due to inefficient eradication strategies and lack of effective vaccines. ALV-J attachment and entry are mediated through the specific receptor, chicken Na+/H+ exchanger type 1 (chNHE1). The non-conserved amino acid tryptophan 38 (W38) in chNHE1 is crucial for virus entry, making it a favorable target for the introduction of disease resistance. In this study, we obtained ALV-J-resistance in a commercial chicken line by precise deletion of chNHE1 W38, utilizing the CRISPR/Cas9-system in combination with homology directed repair. The genetic modification completely protected cells from infection with a subgroup J retrovirus. W38 deletion did neither have a negative effect on the development nor on the general health condition of the gene edited chickens. Overall, the generation of ALV-J-resistant birds by precise gene editing demonstrates the immense potential of this approach as an alternative disease control strategy in poultry