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.
Highlights
The chicken embryo is a key model system in developmental biology [1]
To test whether clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 could be used to edit the primordial germ cells (PGCs) genome, we first performed an experiment to inactivate an enhanced green fluorescent protein (EGFP) transgene inserted in the IgH locus
PGCs were transiently transfected with constructs encoding Cas9 with and without gRNA5
Summary
The chicken embryo is a key model system in developmental biology [1]. Fundamental contributions have been made in the areas of immunology, neurobiology, pattern formation, and others, taking advantage of the ease of access to the embryo in the egg. Despite the importance of chickens in research, the ability to produce birds carrying targeted genetic modifications such as knockouts and knock-ins for functional studies in whole animals has only recently become available [2]. Genome modification in chickens has been accomplished using germline stem cells, such as primordial germ cells (PGCs) or gonocytes, as intermediates [3,4,5,6,7]. Cultured PGCs can be transfected and injected into embryos, where they enter the germline, to obtain gene knockouts with standard homologous recombination vectors [8]. Targeting efficiencies of ~30% can be expected when using homology regions of about 7-8kb. The targeting efficiency is high, obtaining these long, isogenic homology regions can be labor-intensive. Gene editing is more accessible and quicker
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