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

Characterization of a Novel Viral Interleukin 8 (vIL-8) Splice Variant Encoded by Marek’s Disease Virus

Marek’s disease virus (MDV) is a highly cell-associated oncogenic alphaherpesvirus that causes lymphomas in various organs in chickens. Like other herpesviruses, MDV has a large and complex double-stranded DNA genome. A number of viral transcripts are generated by alternative splicing, a process that drastically extends the coding capacity of the MDV genome. One of the spliced genes encoded by MDV is the viral interleukin 8 (vIL-8), a CXC chemokine that facilitates the recruitment of MDV target cells and thereby plays an important role in MDV pathogenesis and tumorigenesis. We recently identified a novel vIL-8 exon (vIL-8-E3′) by RNA-seq; however, it remained elusive whether the protein containing the vIL-8-E3′ is expressed and what role it may play in MDV replication and/or pathogenesis. To address these questions, we first generated recombinant MDV harboring a tag that allows identification of the spliced vIL-8-E3′ protein, revealing that it is indeed expressed. We subsequently generated knockout viruses and could demonstrate that the vIL-8-E3′ protein is dispensable for MDV replication as well as secretion of the functional vIL-8 chemokine. Finally, infection of chickens with this vIL-8-E3′ knockout virus revealed that the protein is not important for MDV replication and pathogenesis in vivo. Taken together, our study provides novel insights into the splice forms of the CXC chemokine of this highly oncogenic alphaherpesvirus

Characterization of a Novel Viral Interleukin 8 (vIL-8) Splice Variant Encoded by Marek’s Disease Virus

Marek’s disease virus (MDV) is a highly cell-associated oncogenic alphaherpesvirus that causes lymphomas in various organs in chickens. Like other herpesviruses, MDV has a large and complex double-stranded DNA genome. A number of viral transcripts are generated by alternative splicing, a process that drastically extends the coding capacity of the MDV genome. One of the spliced genes encoded by MDV is the viral interleukin 8 (vIL-8), a CXC chemokine that facilitates the recruitment of MDV target cells and thereby plays an important role in MDV pathogenesis and tumorigenesis. We recently identified a novel vIL-8 exon (vIL-8-E3′) by RNA-seq; however, it remained elusive whether the protein containing the vIL-8-E3′ is expressed and what role it may play in MDV replication and/or pathogenesis. To address these questions, we first generated recombinant MDV harboring a tag that allows identification of the spliced vIL-8-E3′ protein, revealing that it is indeed expressed. We subsequently generated knockout viruses and could demonstrate that the vIL-8-E3′ protein is dispensable for MDV replication as well as secretion of the functional vIL-8 chemokine. Finally, infection of chickens with this vIL-8-E3′ knockout virus revealed that the protein is not important for MDV replication and pathogenesis in vivo. Taken together, our study provides novel insights into the splice forms of the CXC chemokine of this highly oncogenic alphaherpesvirus

Abrogation of Marek’s disease virus replication using CRISPR/Cas9

Marek’s disease virus (MDV) is a highly cell-associated alphaherpesvirus that causes deadly lymphomas in chickens. While vaccination protects against clinical symptoms, MDV field strains can still circulate in vaccinated flocks and continuously evolve towards greater virulence. MDV vaccines do not provide sterilizing immunity, allowing the virus to overcome vaccine protection, and has increased the need for more potent vaccines or alternative interventions. In this study, we addressed if the CRISPR/Cas9 system can protect cells from MDV replication. We first screened a number of guide RNAs (gRNAs) targeting essential MDV genes for their ability to prevent virus replication. Single gRNAs significantly inhibited virus replication, but could result in the emergence of escape mutants. Strikingly, combining two or more gRNAs completely abrogated virus replication and no escape mutants were observed upon serial passaging. Our study provides the first proof-of-concept, demonstrating that the CRISPR/Cas9 system can be efficiently used to block MDV replication. The presented findings lay the foundation for future research to completely protect chickens from this deadly pathogen