Direct delivery of adenoviral CRISPR/Cas9 vector into the blastoderm for generation of targeted gene knockout in quail

Abstract

Zygotes at the 1-cell stage have been genetically modified by microinjecting the CRISPR/Cas9 components for the generation of targeted gene knockout in mammals. In the avian species, genetic modification of the zygote is difficult because its unique reproductive system limits the accessibility of the zygote at the 1-cell stage. To date, only a few CRISPR/Cas9-mediated gene knockouts have been reported using the chicken as a model among avian species, which requires 3 major processes: isolation and culture of primordial germ cells (PGCs), modification of the genome of PGCs in vitro, and injection of the PGCs into the extraembryonic blood vessel at the early embryonic stages when endogenous PGCs migrate through circulation to the genital ridge. In the present study, the adenoviral CRISPR/Cas9 vector was directly injected into the quail blastoderm in newly laid eggs. The resulting chimeras generated offspring with targeted mutations in the melanophilin (MLPH) gene, which is involved in melanosome transportation and feather pigmentation. MLPH homozygous mutant quail exhibited gray plumage, whereas MLPH heterozygous mutants and wild-type quail exhibited dark brown plumage. In addition, the adenoviral vector was not integrated into the genome of knockout quail, and no mutations were detected in potential off-target regions. This method of generating genome-edited poultry is expected to accelerate avian research and has potential applications for developing superior genetic lines for poultry production in the industry.

Keywords: CRISPR/Cas9; MLPH; adenovirus; genome editing; poultry.

Fig. 1.

Generation of targeted gene knockout quail using adenovirus. (A) Schematic representation of research plan. gRNA of the MLPH gene was selected from exon 2, and the adenoviral CRISPR/Cas9 vector was constructed. Recombinant adenovirus was subsequently produced and injected into the quail blastoderm. Chimeras (G0) were maintained and mated with wild-type (WT) quail to produce G1 quail with a heterozygous genotype of the MLPH gene (MLPH^+/−). Male and female G1 MLPH^+/− quail were subsequently mated to generate G2 quail with a homozygous genotype of the MLPH gene (MLPH^−/−). After production of the recombinant adenovirus, it took a total of 10 wk, 2 wk for egg incubation after virus injection and another 8 wk for sexual maturation, to receive eggs from G0 quail. After G1 quail hatched, the MLPH^+/− quail were screened and mated to produce G2 offspring 8 wk later. Fully grown G2 offspring were obtained for further experiments 26 wk after the injection of the adenovirus. (B) Sanger sequencing chromatograms of G1 MLPH^+/− quail. Dashed lines indicate the starting point of the mutation, and the deleted or inserted nucleotides are underlined. The PAM sequences are highlighted in gray. Nucleotide sequences are presented in a negative direction.

Fig. 2.

Phenotypic and genotypic comparison of G2 offspring. (Left) WT and MLPH^+/− quail with dark brown plumage and MLPH^−/− quail with gray plumage without any change in plumage pattern. (Right) DNA sequencing chromatograms of MLPH^+/− and MLPH^−/− quail, which indicate there is a one base pair deletion of one allele and both alleles, respectively. Dashed line indicates the point where one base pair deletion occurred. The PAM sequences are highlighted in gray. Nucleotide sequences are presented in a negative direction.