title: Efficient gene targeting in golden Syrian hamsters by the CRISPR/Cas9 system.
abstract: The golden Syrian hamster is the model of choice or the only rodent model for studying many human diseases. However, the lack of gene targeting tools in hamsters severely limits their use in biomedical research. Here, we report the first successful application of the CRISPR/Cas9 system to efficiently conduct gene targeting in hamsters. We designed five synthetic single-guide RNAs (sgRNAs)--three for targeting the coding sequences for different functional domains of the hamster STAT2 protein, one for KCNQ1, and one for PPP1R12C--and demonstrated that the CRISPR/Cas9 system is highly efficient in introducing site-specific mutations in hamster somatic cells. We then developed unique pronuclear (PN) and cytoplasmic injection protocols in hamsters and produced STAT2 knockout (KO) hamsters by injecting the sgRNA/Cas9, either in the form of plasmid or mRNA, targeting exon 4 of hamster STAT2. Among the produced hamsters, 14.3% and 88.9% harbored germline-transmitted STAT2 mutations from plasmid and mRNA injection, respectively. Notably, 10.4% of the animals produced from mRNA injection were biallelically targeted. This is the first success in conducting site-specific gene targeting in hamsters and can serve as the foundation for developing other genetically engineered hamster models for human disease.
authors.affiliation: Department of Animal, Dairy, and Veterinary Sciences, Utah State University, Logan, Utah, United States of America.
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Fan
authors.affiliation: Department of Animal, Dairy, and Veterinary Sciences, Utah State University, Logan, Utah, United States of America.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.affiliation: Department of Animal, Dairy, and Veterinary Sciences, Utah State University, Logan, Utah, United States of America.
authors.forename: Sang R
authors.initials: SR
authors.lastname: Lee
authors.affiliation: Department of Animal, Dairy, and Veterinary Sciences, Utah State University, Logan, Utah, United States of America.
authors.forename: Qinggang
authors.initials: Q
authors.lastname: Meng
authors.affiliation: Department of Animal, Dairy, and Veterinary Sciences, Utah State University, Logan, Utah, United States of America.
authors.forename: Bi
authors.initials: B
authors.lastname: Shi
authors.affiliation: Department of Animal, Dairy, and Veterinary Sciences, Utah State University, Logan, Utah, United States of America.
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Bunch
authors.affiliation: Department of Animal, Dairy, and Veterinary Sciences, Utah State University, Logan, Utah, United States of America.
authors.forename: Kenneth L
authors.initials: KL
authors.lastname: White
authors.affiliation: Department of Animal Science, Division of Applied Life Science, Gyeongsang National University, Jinju, Gyeongnam Province, Republic of Korea; Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, Gyeongnam Province, Republic of Korea.
authors.forename: Il-Keun
authors.initials: IK
authors.lastname: Kong
authors.affiliation: Department of Animal, Dairy, and Veterinary Sciences, Utah State University, Logan, Utah, United States of America; Auratus Bio, LLC., Canton, South Dakota, United States of America.
authors.forename: Zhongde
authors.initials: Z
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D000076987:CRISPR-Associated Protein 9; D064130:CRISPR-Associated Proteins / Q000235:genetics* / Q000378:metabolism; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods; D051657:KCNQ1 Potassium Channel / Q000235:genetics / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008647:Mesocricetus / Q000235:genetics*; D008845:Microinjections; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics; D054645:Protein Phosphatase 1 / Q000235:genetics / Q000378:metabolism; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000008:administration & dosage / Q000235:genetics; D050795:STAT2 Transcription Factor / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D064130:CRISPR-Associated Proteins; D051657:KCNQ1 Potassium Channel; D000094704:RNA, Guide, CRISPR-Cas Systems; D012333:RNA, Messenger; D050795:STAT2 Transcription Factor; D000076987:CRISPR-Associated Protein 9; C000600049:Cas9 protein, Francisella novicida; D004720:Endonucleases; D054645:Protein Phosphatase 1