Most cited article - PubMed ID 29079843
Male fertility restored by transplanting primordial germ cells into testes: a new way towards efficient transgenesis in chicken
The chicken Tva cell surface protein, a member of the low-density lipoprotein receptor family, has been identified as an entry receptor for avian leukosis virus of classic subgroup A and newly emerging subgroup K. Because both viruses represent an important concern for the poultry industry, we introduced a frame-shifting deletion into the chicken tva locus with the aim of knocking-out Tva expression and creating a virus-resistant chicken line. The tva knock-out was prepared by CRISPR/Cas9 gene editing in chicken primordial germ cells and orthotopic transplantation of edited cells into the testes of sterilized recipient roosters. The resulting tva -/- chickens tested fully resistant to avian leukosis virus subgroups A and K, both in in vitro and in vivo assays, in contrast to their susceptible tva +/+ and tva +/- siblings. We also found a specific disorder of the cobalamin/vitamin B12 metabolism in the tva knock-out chickens, which is in accordance with the recently recognized physiological function of Tva as a receptor for cobalamin in complex with transcobalamin transporter. Last but not least, we bring a new example of the de novo resistance created by CRISPR/Cas9 editing of pathogen dependence genes in farm animals and, furthermore, a new example of gene editing in chicken.
- Keywords
- avian leukosis virus subgroups A/K, gene editing in chicken, tva, vitamin B12/cobalamin,
- MeSH
- Gene Editing MeSH
- Gene Knockout Techniques MeSH
- Chickens virology MeSH
- Chick Embryo MeSH
- Methylmalonic Acid blood MeSH
- Frameshift Mutation MeSH
- Avian Proteins genetics physiology MeSH
- Receptors, Virus genetics physiology MeSH
- Avian Leukosis Virus classification physiology MeSH
- Vitamin B 12 metabolism MeSH
- Animals MeSH
- Check Tag
- Chick Embryo MeSH
- Male MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Methylmalonic Acid MeSH
- Avian Proteins MeSH
- Tva receptor MeSH Browser
- Receptors, Virus MeSH
- Vitamin B 12 MeSH
Avian leukosis virus subgroup J (ALV-J) is an important concern for the poultry industry. Replication of ALV-J depends on a functional cellular receptor, the chicken Na+/H+ exchanger type 1 (chNHE1). Tryptophan residue number 38 of chNHE1 (W38) in the extracellular portion of this molecule is a critical amino acid for virus entry. We describe a CRISPR/Cas9-mediated deletion of W38 in chicken primordial germ cells and the successful production of the gene-edited birds. The resistance to ALV-J was examined both in vitro and in vivo, and the ΔW38 homozygous chickens tested ALV-J-resistant, in contrast to ΔW38 heterozygotes and wild-type birds, which were ALV-J-susceptible. Deletion of W38 did not manifest any visible side effect. Our data clearly demonstrate the antiviral resistance conferred by precise CRISPR/Cas9 gene editing in the chicken. Furthermore, our highly efficient CRISPR/Cas9 gene editing in primordial germ cells represents a substantial addition to genotechnology in the chicken, an important food source and research model.
- Keywords
- CRISPR/Cas9 genome editing in chicken, Na+/H+ exchanger type 1, avian leukosis virus subgroup J, disease resilience in poultry, primordial germ cells,
- MeSH
- CRISPR-Cas Systems MeSH
- Gene Editing MeSH
- Animals, Genetically Modified genetics immunology virology MeSH
- Chickens MeSH
- Poultry Diseases genetics immunology virology MeSH
- Disease Resistance MeSH
- Avian Leukosis genetics immunology virology MeSH
- Avian Proteins genetics immunology MeSH
- Sodium-Hydrogen Exchanger 1 genetics immunology MeSH
- Avian Leukosis Virus classification genetics physiology MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Avian Proteins MeSH
- Sodium-Hydrogen Exchanger 1 MeSH
It has now been more than two years since we said our last goodbye to Jan Svoboda (14 [...].
Avian leukosis viruses (ALVs), which are pathogens of concern in domestic poultry, utilize specific receptor proteins for cell entry that are both necessary and sufficient for host susceptibility to a given ALV subgroup. This unequivocal relationship offers receptors as suitable targets of selection and biotechnological manipulation with the aim of obtaining virus-resistant poultry. This approach is further supported by the existence of natural knock-outs of receptor genes that segregate in inbred lines of chickens. We used CRISPR/Cas9 genome editing tools to introduce frame-shifting indel mutations into tva, tvc, and tvj loci encoding receptors for the A, C, and J ALV subgroups, respectively. For all three loci, the homozygous frame-shifting indels generating premature stop codons induced phenotypes which were fully resistant to the virus of respective subgroup. In the tvj locus, we also obtained in-frame deletions corroborating the importance of W38 and the four amino-acids preceding it. We demonstrate that CRISPR/Cas9-mediated knock-out or the fine editing of ALV receptor genes might be the first step in the development of virus-resistant chickens.
- Keywords
- CRISPR/Cas9, avian leukosis virus, retrovirus receptor, virus-resistance in chicken,
- MeSH
- Cell Line MeSH
- CRISPR-Cas Systems * MeSH
- Gene Editing * MeSH
- Genetic Techniques MeSH
- Genetic Vectors genetics MeSH
- Chickens MeSH
- Disease Resistance genetics MeSH
- Avian Leukosis genetics virology MeSH
- Base Sequence MeSH
- Genes, Viral MeSH
- Receptors, Virus genetics metabolism MeSH
- Avian Leukosis Virus physiology MeSH
- RNA, Guide, CRISPR-Cas Systems MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Receptors, Virus MeSH
- RNA, Guide, CRISPR-Cas Systems MeSH
