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.

Mapping the Specific Amino Acid Residues That Make Hamster DPP4 Functional as a Receptor for Middle East Respiratory Syndrome Coronavirus

“…Descriptions of transgenic hamsters are virtually absent from the scientific literature due to the absence of specific gene-targeting tools. Utilization of the clustered regularly interspaced short palindromic repeat (CRISPR)/ Cas9 system would allow efficient gene targeting and the generation of a new small-animal model (9,10). Hamster a hpi, hours postinfection; boldface, human residues; italics, hamster residues; ϩ/ϩ, viral growth; ϩ/Ϫ, attenuated viral growth; Ϫ/Ϫ, no viral growth; ϽDL, below the detection limit of the assay.…”

“…Therefore, if a treatment against MERS-CoV is shown to be successful in the mouse model, further characterization of the treatment needs to be performed in NHPs, a relatively expensive animal model to which access is limited. The availability of a second small-animal model (such as hamsters with a modified DPP4 [9,10]) to confirm results obtained with the mouse model would ensure that only treatments with a high likelihood of succeeding would be investigated in NHPs.Fourteen amino acids are important in the interaction between blades IV and V of human DPP4 (hDPP4) and the receptor binding domain (RBD) of the MERS-CoV spike protein (11,12). We previously showed that hamster DPP4 (haDPP4) does not function as a receptor for MERS-CoV.…”

“…Therefore, if a treatment against MERS-CoV is shown to be successful in the mouse model, further characterization of the treatment needs to be performed in NHPs, a relatively expensive animal model to which access is limited. The availability of a second small-animal model (such as hamsters with a modified DPP4 [9,10]) to confirm results obtained with the mouse model would ensure that only treatments with a high likelihood of succeeding would be investigated in NHPs.…”

Modeling Severe Fever with Thrombocytopenia Syndrome Virus Infection in Golden Syrian Hamsters: Importance of STAT2 in Preventing Disease and Effective Treatment with Favipiravir

“…The development of the STAT2 KO golden Syrian hamsters used has been previously described (20). Male and female 6-to 7-week-old STAT2 KO hamsters were obtained from the breeding colony at Utah State University (Logan, UT).…”

Heartland virus infection in hamsters deficient in type I interferon signaling: Protracted disease course ameliorated by favipiravir

“…The development of the STAT2 KO golden Syrian hamsters used has been previously described (Fan et al, 2014). Male and female STAT2 KO hamsters (4–5 weeks of age) were used for all experiments and obtained from the breeding colony at Utah State University (USU).…”