Targeted mutagenesis in chicken using CRISPR/Cas9 system

www.nature.com/scientificreports OPEN Targeted mutagenesis in chicken using CRISPR/Cas9 system Isao Oishi1, Kyoko Yoshii1, Daichi Miyahara2, Hiroshi Kagami2 & Takahiro Tagami3 received: 23 October 2015 accepted: 17 March 2016 Published: 06 April 2016 The CRISPR/Cas9 system is a simple and powerful tool for genome editing in various organisms including livestock animals. However, the system has not been applied to poultry because of the difficulty in accessing their zygotes. Here we report the implementation of CRISPR/Cas9-mediated gene targeting in chickens. Two egg white genes, ovalbumin and ovomucoid, were efficiently (>90%) mutagenized in cultured chicken primordial germ cells (PGCs) by transfection of circular plasmids encoding Cas9, a single guide RNA, and a gene encoding drug resistance, followed by transient antibiotic selection. We transplanted CRISPR-induced mutant-ovomucoid PGCs into recipient chicken embryos and established three germline chimeric roosters (G0). All of the roosters had donor-derived mutant-ovomucoid spermatozoa, and the two with a high transmission rate of donor-derived gametes produced heterozygous mutant ovomucoid chickens as about half of their donor-derived offspring in the next generation (G1). Furthermore, we generated ovomucoid homozygous mutant offspring (G2) by crossing the G1 mutant chickens. Taken together, these results demonstrate that the CRISPR/Cas9 system is a simple and effective gene-targeting method in chickens. Chicken is a commercially important animal and its genetic modification is expected to be used for agricultural, industrial, and scientific applications1–3. There are various possible and beneficial applications of genetically modified chicken, including improvement of production of meat and eggs, generation of disease-resistant chickens, mass production of therapeutic proteins in egg whites, and establishment of models for studying avian development. Furthermore, gene disruption of egg white allergen genes such as ovalbumin (OVA) and ovomucoid (OVM) has the potential to produce low allergenicity in eggs, thereby reducing immune responses in individuals sensitive to items such as egg white–containing food products and vaccines. To produce these genetically modified chickens, efficient technologies, including suitable transgenic and knockout methods, are required. However, the genetic modification of chickens has lagged far behind that of other organisms because of the difficulty in accessing and manipulating the zygote4. Therefore, chicken transgenesis has mainly been performed using viral vector infection of the early stage embryo5,6. Recently, primordial germ cells (PGCs), which can be cultured and genetically modified in vitro, have been used to generate transgenic chickens by taking advantage of their germline competency after injection into recipient embryos7–11. In contrast to dozens of studies generating transgenic chickens with these methods, few reports are available on chicken gene disruption. The generation of knockout chickens was first reported by Schusser et al.12. In this study, the immunoglobulin heavy chain gene was disrupted in cultured PGCs by homologous recombination with a targeting construct. Thereafter, heterozygous and homozygous gene-targeted chickens were produced in G1 and G2 offspring of germline chimeric chickens that had been transplanted with the gene-disrupted PGCs. Recently, Park et al. reported OVA heterozygous knockout chicken produced by transcription activator-like effector nuclease (TALEN)-induced PGC mutation13. This study was the first to demonstrate the effectiveness of site-specific nuclease-mediated genome-editing technology in the generation of mutant chickens. Another genome-editing technology is the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein system, known as CRISPR/Cas14,15. CRISPR/Cas9 uses an RNA-guided nuclease (Cas9) to target specific sequences and induces DNA double-stranded breaks (DSBs) therein. During the DSB repair process by non-homologous end-joining (NHEJ), small insertions or deletions (indels) are efficiently introduced, and the indels can lead to shifts in the reading frame and the ultimate functional disruption of targeted proteins. Because the CRISPR/Cas9 system requires only a pair of oligonucleotides containing the target sequence, 1 Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, 1-8-31, Midorioka, Ikeda, Osaka 563-8577, Japan. 2Faculty of Agriculture, Shinshu University, 8304 Minamiminowa, Nagano 399-4598, Japan. 3Animal Breeding and Reproduction Research Division, National Agriculture and Food Research Organization, Institute of Livestock and Grassland Science, 2 Ikenodai, Tsukuba, Ibaraki 305-0901, Japan. Correspondence and requests for materials should be addressed to I.O. (email: ) or T.T. (email: ) Scientific Reports | 6:23980 | DOI: 10.1038/srep23980 1 www.nature.com/scientificreports/ Figure 1. Schematic representation of sgRNAs targeting the OVA and OVM loci. Exon-intron organization and targeting sequences for the OVA (upper) and OVM (lower) loci are shown. DNA and amino acid sequences are shown in lowercase and uppercase letters, respectively, and correspond to the regions indicated by the red arrows. The four sgRNA targeting sites are numbered and represented by black bars above the nucleotide sequence. Adjoining protospacer adjacent motif (PAM) sequences are highlighted in red. The OVMTg1 target site spans the intron 2/exon 3 boundary. preparation of the plasmid for targeted gene disruption is much easier and more cost-effective compared with the TALEN-mediated method14,15. To date, various organisms have been mutagenized using the CRISPR/Cas9 system16, including livestock animals such as pig17, rabbit18, and goat19, but not avian species. Therefore, the methods should be developed to apply the CRISPR/Cas9 system to generate gene targeting in chickens. Here we report successful gene targeting of OVM in the chicken using the CRISPR/Cas9 system. A single plasmid transfection followed by antibiotic selection resulted in targeted mutation with > 90% efficiency in chicken PGCs. The mutated PGCs generated functional gametes via germline chimera and produced male and female G1 offspring with various OVM mutations. In addition, OVM−/− mutant chickens were obtained as G2 offspring by crossing OVM+/− mutant chickens. Results Validation of single guide RNA (sgRNA) disruption of OVA and OVM. To use the CRISPR/Cas9 system for targeted mutagenesis in chickens, which will lead to the improvement of chicken products, we chose two egg white genes, ovalbumin (OVA) and ovomucoid (OVM), as targets for disruption. Four targeting (Tg) sites close to the initiation codon were selected for each gene: OVATg1–4 in exon 2 of OVA, and OVMTg1–4 in exon 3 of OVM, and sgRNAs were designed for each target sequence (Fig. 1). Eight neomycin resistance–based plasmids (...truncated)