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

Abstract

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.

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

“…They are used for studying neurological and cardiovascular functions ( 6 – 8 ) and provided key findings in B cell development and graft versus host responses ( 9 – 11 ). Genetically modified livestock species also hold great promise for agriculture by offering new approaches for disease control, such as genome-edited pigs resistant to Porcine Reproductive and Respiratory Syndrome or Avian Leucosis Virus (ALV)-resistant chickens ( 12 – 15 ).…”

A Genetically Engineered Commercial Chicken Line Is Resistant to Highly Pathogenic Avian Leukosis Virus Subgroup J

“…The genetically modified chicken line was established recently [ 15 ]. The genetic background of these birds is a widely used, commercial egg-type chicken line (White Leghorn line; Lohmann selected Leghorn) obtained from Lohmann-Tierzucht GmbH (Cuxhaven, Germany).…”

“…Here, CRISPR/Cas9-based genome editing serves as a precise and efficient molecular tool to modify host genes that play a key role in the viral replication cycles and render the host resistant against the pathogen [ 12 , 13 ]. We and others recently used this strategy to generate ALV-J resistant chickens [ 14 , 15 ]. Virus entry was inhibited by a precise modification of the ALV-J host cell receptor, chicken Na + /H + exchanger type 1 (chNHE1), encoded by the tvj locus on chromosome 23 [ 16 ].…”

“…The crucial role of a single amino acid, the non-conserved tryptophan 38 (W38) for ALV-J binding, has been demonstrated previously in vitro [ 8 , 18 , 19 ]. Based on these findings, the chNHE1 modification in the generated chicken line consists of a single amino acid deletion of tryptophan 38 (W38) [ 14 , 15 ]. In both studies, ALV-J resistance was assessed utilizing a GFP-transducing [ 14 , 15 ] or a v- src -transducing [ 14 ] subgroup-J specific RCAS (replication-competent avian sarcoma-leukosis virus with a splice acceptor) reporter virus.…”

“…Based on these findings, the chNHE1 modification in the generated chicken line consists of a single amino acid deletion of tryptophan 38 (W38) [ 14 , 15 ]. In both studies, ALV-J resistance was assessed utilizing a GFP-transducing [ 14 , 15 ] or a v- src -transducing [ 14 ] subgroup-J specific RCAS (replication-competent avian sarcoma-leukosis virus with a splice acceptor) reporter virus. While this is a valuable system to evaluate ALV-J susceptibility, it does not fully represent field conditions [ 18 ].…”