Animal Molecular Breeding, 2024, Vol.14, No.6, 345-353 http://animalscipublisher.com/index.php/amb 349 to its impact on chicken health and productivity. The study selected chickens as the model species due to their importance in the poultry industry and the feasibility of genetic modifications in this species (Oishi et al., 2016). 6.2 Implementation of CRISPR/Cas9 for disease resistance The implementation of CRISPR/Cas9 in this case involved precise editing of the chicken Na+/H+ exchanger type 1 (chNHE1) gene, which encodes a receptor critical for ALV-J entry into chicken cells. Researchers introduced a single amino acid deletion at tryptophan residue number 38 (W38) in the chNHE1 gene. This specific mutation was chosen because W38 is essential for the virus's ability to infect chicken cells (Figure 2) (Koslová et al., 2020). Figure 2 Design of guide RNA and homologous recombination ssODN for CRISPR/Cas9 gene editing of chicken ALV-J receptor chNHE1in primordial germ cells (Adopted from Koslová et al., 2020) Image caption: (A) The structure of coding exons and introns of chNHE1(Top), the CRISPR/Cas9 target sequence of exon 1 with the guide RNA (gRNA) complementary sequence (underlined) and the TGG triplet encoding W38 (red on the yellow background; Middle), and the central part of the ssODN template for homologous recombination with deleted TGG triplet and a single nucleotide substitution (in red) creating the BsaI restriction site (Bottom). (B) Preparation of ΔW38 chickens: schematic representation of the workflow and timeline (Adopted from Koslová et al., 2020) The CRISPR/Cas9 system was used to delete the W38 residue in chicken primordial germ cells (PGCs). These edited PGCs were then used to produce gene-edited chickens. The resulting ΔW38 homozygous chickens were tested for resistance to ALV-J both in vitro and in vivo. The results showed that ΔW38 homozygous chickens were resistant to ALV-J, whereas ΔW38 heterozygotes and wild-type birds remained susceptible (Table 1) (Koslová et al., 2020). Table 1 ALV-J viremia in chickens inoculated with RCASBP(J)GFP (Adopted from Koslová et al., 2020) chNHE1 genotype Chicken no. Age of chicken, d Virus titer, IU/mL 6dp.i. 13dp.i. W38−/− 604 28 0 0 W38−/− 606 28 0 0 W38−/− 611 20 0 0 W38−/− 612 20 0 0 W38−/− 615 14 0 0 W38+/− 601 28 0 101 W38+/− 603 28 0 102 W38+/− 605 28 0 101 W38+/− 610 20 102 103 W38+/− 616 20 0 103 W38+/− 618 14 0 102 W38+/− 619 14 101 103 W38+/+ 617 14 102 103
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