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

• Published in: PloS one Vol. 11; no. 4; p. e0154303
• Main Authors: Dimitrov, Lazar, Pedersen, Darlene, Ching, Kathryn H, Yi, Henry, Collarini, Ellen J, Izquierdo, Shelley, van de Lavoir, Marie-Cecile, Leighton, Philip A
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 21.04.2016; Public Library of Science (PLoS)
• Subjects: Animal tissues; Animals; Animals, Genetically Modified; Base Sequence; Biology and Life Sciences; Birds; Chickens; Chickens - genetics; Chickens - growth & development; Cloning; Cloning, Organism; CRISPR; CRISPR-Cas Systems; Deoxyribonucleic acid; DNA; Double-strand break repair; Drug resistance; Embryo, Nonmammalian; Engineering; Engineering and Technology; Female; Flow cytometry; Gene Editing - methods; Gene Knockout Techniques; Genetic aspects; Genetic engineering; Genetic modification; Genetic Vectors - chemistry; Genetic Vectors - metabolism; Genome; Genome editing; Genomes; Germ Cells; Green Fluorescent Proteins - deficiency; Green Fluorescent Proteins - genetics; Homologous Recombination; Homology; Immunoglobulin Heavy Chains - genetics; Immunoglobulins; Loci; Male; Methods; Mutation; Neurosciences; Nucleases; Offspring; Physiological aspects; Poultry; Progeny; Research and Analysis Methods; RNA, Guide, CRISPR-Cas Systems - genetics; RNA, Guide, CRISPR-Cas Systems - metabolism; Rodents; Stem cells; Transgenic; United States; United States > US; California
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0154303

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.