Targeted mutagenesis in chicken using CRISPR/Cas9 system.

Area of Science:

• Genetics
• Molecular Biology
• Animal Science

Background:

• The CRISPR/Cas9 system offers powerful genome editing capabilities across species.
• Application in poultry was limited due to challenges in accessing zygotes for gene editing.

Purpose of the Study:

• To implement and validate CRISPR/Cas9-mediated gene targeting in chickens.
• To demonstrate the efficiency of gene editing in chicken primordial germ cells (PGCs).

Main Methods:

• Cultured chicken primordial germ cells (PGCs) were transfected with plasmids encoding Cas9 and single guide RNA.
• Genes for ovalbumin and ovomucoid were targeted for mutagenesis (>90% efficiency).
• CRISPR-induced mutant PGCs were transplanted into recipient embryos, establishing germline chimeras.

Main Results:

• High-efficiency mutagenesis (>90%) of ovalbumin and ovomucoid genes in chicken PGCs.
• Successful generation of germline chimeric roosters (G0) with mutant ovomucoid spermatozoa.
• Transmission of the ovomucoid mutation to offspring (G1) and generation of homozygous mutants (G2).

Conclusions:

• CRISPR/Cas9 is an effective and simple gene-targeting method for chickens.
• This study overcomes previous limitations in applying genome editing to poultry.
• The findings open new avenues for genetic modification in avian species.