Efficient gene targeting in golden Syrian hamsters by the CRISPR/Cas9 system

Zhiqiang Fan; Wei Li; Sang R Lee; Qinggang Meng; Bi Shi; Thomas D Bunch; Kenneth L White; Il-Keun Kong; Zhongde Wang

doi:10.1371/journal.pone.0109755

Efficient gene targeting in golden Syrian hamsters by the CRISPR/Cas9 system

PLoS One. 2014 Oct 9;9(10):e109755. doi: 10.1371/journal.pone.0109755. eCollection 2014.

Authors

Zhiqiang Fan¹, Wei Li¹, Sang R Lee¹, Qinggang Meng¹, Bi Shi¹, Thomas D Bunch¹, Kenneth L White¹, Il-Keun Kong², Zhongde Wang³

Affiliations

¹ Department of Animal, Dairy, and Veterinary Sciences, Utah State University, Logan, Utah, United States of America.
² 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.
³ 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.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Animals
Animals, Genetically Modified
Bacterial Proteins / genetics*
Bacterial Proteins / metabolism
CRISPR-Associated Protein 9
CRISPR-Associated Proteins / genetics*
CRISPR-Associated Proteins / metabolism
Cell Nucleus / genetics
Cell Nucleus / metabolism
Cytosol / metabolism
Endonucleases / genetics*
Endonucleases / metabolism
Epithelial Cells / cytology
Epithelial Cells / metabolism
Gene Targeting / methods*
Genetic Engineering / methods
KCNQ1 Potassium Channel / genetics
KCNQ1 Potassium Channel / metabolism
Kidney / cytology
Kidney / metabolism
Mesocricetus / genetics*
Microinjections
Molecular Sequence Data
Mutagenesis, Site-Directed
Plasmids / administration & dosage
Plasmids / genetics
Protein Phosphatase 1 / genetics
Protein Phosphatase 1 / metabolism
RNA, Guide, CRISPR-Cas Systems / genetics
RNA, Guide, CRISPR-Cas Systems / metabolism
RNA, Messenger / administration & dosage
RNA, Messenger / genetics
STAT2 Transcription Factor / genetics
STAT2 Transcription Factor / metabolism

Substances

Bacterial Proteins
CRISPR-Associated Proteins
KCNQ1 Potassium Channel
RNA, Guide, CRISPR-Cas Systems
RNA, Messenger
STAT2 Transcription Factor
CRISPR-Associated Protein 9
Cas9 protein, Francisella novicida
Endonucleases
Protein Phosphatase 1

Grants and funding

This work was supported by the Utah Science Technology and Research (USTAR) initiative (to ZW); and the Next-Generation Bio-Green 21 Program (No. PJ009587022013) by the Rural Development Administration (RDA) and BK21 plus program, Republic of Korea (to IKK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.