Utilization of the CRISPR/Cas9 systems to protect chickens against Mareks disease virus and as a tool to dissect virus pathogenesis
Final Report Abstract
The chicken is an excellent model in developmental biology as well as many other research areas. Beyond that, they are the most important source of animal protein for humans and secures the livelihood of millions of people. A plethora of pathogens threaten the welfare and life of billions of chickens. One of these pathogens is Marek’s disease virus (MDV) that causes immunosuppression and deadly lymphomas in susceptible chickens. Despite vaccination that protects against the deadly symptoms, MDV can circulate in vaccinated flocks and continuously develops toward a higher virulence. In the past, this allowed the virus to overcome the vaccine protection, increasing the need for more potent vaccines or other interventions. Bacteria use the flexible CRISPR/Cas9 system to defend themselves from invading viruses, which is now widely used for gene editing. Therefore, we hypothesized that this CRISPR/Cas9 system can be used to protect chickens from deadly pathogens such as MDV and that this system will also help us to investigate cellular factors involved in MDV-induced tumor development. We addressed this hypothesis in two specific aims by i) transferring the bacterial immune system CRISPR/Cas9 into chickens to protect them from a deadly MDV challenge and ii) to use CRISPR/Cas9 to characterize the role of cellular CD30 in the development of MDV-induced lymphomas. To achieve aim 1, we generated Cas9 nuclease expressing chickens as a basic tool for avian research. In addition, we screened guide RNAs (gRNAs) for their ability to abrogate MDV replication in cells expressing Cas9 to identify the optimal gRNAs for the insertion into the chicken genome. To obtain chickens with a functional CRISPR/Cas9 targeting MDV, we mated Cas9 birds with chickens expressing the validated MDV specific gRNAs. Even though our Cas9 approach efficiently abrogated MDV replication in vitro2, it did not protect chickens against a deadly MDV challenge. Cas9 expression was substantially lower in chickens compared the cells culture, representing the limiting factor for the protection. In addition to targeting MDV, we successfully used the CRISPR/Cas9 system to generate chickens that are resistant against the highly pathogenic avian leukosis virus subgroup J (ALV-J). To address aim 2, we set to investigate the role of the cellular surface molecule CD30, which is highly expressed in MDV-induced tumors as well as various lymphomas in humans. First, we screened gRNAs against CD30 and identified the ones that facilitate abrogation of CD30 in ESCDL-1 cells. In parallel, we generated recombinant MDV expressing the CRISPR/Cas9 system. Even though Cas9 was initially well expressed, the virus lost its expression over time, likely due to negative selection. To overcome this, we used cells from the Cas9 expressing chickens (aim 1) to confirm the excision of CD30. While excision of CD30 was efficient the Cas9 expressing ESCDL-1 cells, hardy any was detected in the cells from the Cas9 chickens likely due to the lower Cas9 expression. As an alternative approach, we used shRNAs encoded by the virus genome to investigate the role of CD30 in MDV pathogenesis. Taken together, we generated Cas9 expressing chickens that can be widely used in avian research, developed an approach that can abrogate MVD replication in vitro and generated chickens resistant against ALV-J.
Publications
- 2018. Unraveling the role of B cells in the pathogenesis of an oncogenic avian herpesvirus. PNAS
Bertzbach L.D., M. Laparidou, S. Härtle, R.J. Etches, B. Kaspers, B. Schusser, B.B. Kaufer
(See online at https://doi.org/10.1073/pnas.1813964115) - 2020. Abrogation of Marek’s disease virus replication using CRISPR/Cas9. Scientific reports
Hagag I.T., D.J. Wight, D. Bartsch, H. Sid, I. Jordan, L.D. Bertzbach, B. Schusser, B.B. Kaufer
(See online at https://doi.org/10.1038/s41598-020-67951-1) - 2020. Acquiring Resistance Against a Retroviral Infection via CRISPR/Cas9 Targeted Genome Editing in a Commercial Chicken Line. Frontiers in Genome Editing
Hellmich R, H. Sid, K. Lengyel, K. Flisikowski, A. Schlickenrieder, D. Bartsch, T. Thoma, L.D. Bertzbach, B.B. Kaufer, V. Nair, R. Preisinger, B. Schusser
(See online at https://doi.org/10.3389/fgeed.2020.00003) - 2021. A Genetically Engineered Commercial Chicken Line Is Resistant to Highly Pathogenic Avian Leukosis Virus Subgroup J. Microorganisms
Kheimar A., R. Klinger, L.D. Bertzbach, H. Sid, Y. Yu, A.M. Conradie, B. Schade, B. Bohm, R. Preisinger, V. Nair, B.B. Kaufer, B. Schusser
(See online at https://doi.org/10.3390/microorganisms9051066) - 2021. Cas9-expressing chickens and pigs as resources for genome editing in livestock. PNAS
Rieblinger B., H. Sid, D. Duda, T. Bozoglu, R. Klinger, A. Schlickenrieder, K. Lengyel, K. Flisikowski, T. Flisikowska, N. Simm, A. Grodziecki, C. Perleberg, A. Bahr, L. Carrier, M. Kurome, V. Zakhartchenko, B. Kessler, E. Wolf, L. Kettler, H. Luksch, I.T. Hagag, D. Wise, J. Kaufman, B.B. Kaufer, C. Kupatt, A. Schnieke, B. Schusser
(See online at https://doi.org/10.1073/pnas.2022562118)
