Germline Gene Editing in Chickens by Efficient CRISPR-Mediated Homologous Recombination in Primordial Germ Cells

PLoS One. 2016 Apr 21;11(4):e0154303. doi: 10.1371/journal.pone.0154303. eCollection 2016.

Abstract

The CRISPR/Cas9 system has been applied in a large number of animal and plant species for genome editing. In chickens, CRISPR has been used to knockout genes in somatic tissues, but no CRISPR-mediated germline modification has yet been reported. Here we use CRISPR to target the chicken immunoglobulin heavy chain locus in primordial germ cells (PGCs) to produce transgenic progeny. Guide RNAs were co-transfected with a donor vector for homology-directed repair of the double-strand break, and clonal populations were selected. All of the resulting drug-resistant clones contained the correct targeting event. The targeted cells gave rise to healthy progeny containing the CRISPR-targeted locus. The results show that gene-edited chickens can be obtained by modifying PGCs in vitro with the CRISPR/Cas9 system, opening up many potential applications for efficient genetic modification in birds.

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • Base Sequence
  • CRISPR-Cas Systems*
  • Chickens / genetics*
  • Chickens / growth & development
  • Cloning, Organism
  • Embryo, Nonmammalian
  • Female
  • Gene Editing / methods*
  • Gene Knockout Techniques
  • Genetic Vectors / chemistry
  • Genetic Vectors / metabolism
  • Genome*
  • Germ Cells
  • Green Fluorescent Proteins / deficiency
  • Green Fluorescent Proteins / genetics
  • Homologous Recombination*
  • Immunoglobulin Heavy Chains / genetics*
  • Male
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • RNA, Guide, CRISPR-Cas Systems / metabolism

Substances

  • Immunoglobulin Heavy Chains
  • RNA, Guide, CRISPR-Cas Systems
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins

Grants and funding

Crystal Bioscience provided support in the form of salaries for all authors, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.