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

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

doi:10.1371/journal.pone.0109755

Zhiqiang Fan, Sang R Lee + Show 8 more

Open Access

https://doi.org/10.1371/journal.pone.0109755

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Journal: PloS one	Publication Date: Oct 9, 2014
Citations: 102	License type: CC BY 4.0

Affiliation: Utah State University, Gyeongsang National University

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.

Highlights

The CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) system is an RNA-based adaptive immune mechanism to degrade invading plasmids and viruses by bacteria and archaea [1]
To test whether the CRISPR/ Cas9 system is effective in introducing targeted mutations in hamster cells, we transfected each of the single-guide RNAs (sgRNAs)/Cas9 expressing vectors individually into baby hamster kidney (BHK) fibroblasts followed by analyzing the potential gene targeting events by using a PCR-restriction fragment length polymorphism (PCR-RFLP) assay (Figure 1B)
To reveal the nature of the genetic mutations introduced by these sgRNA/Cas9 vectors, we focused on analyzing BHK cells transfected by sgRNA/Cas9STAT2-nd, sgRNA/Cas9-KCNQ1 and sgRNA/Cas9-PPP1R12C

Summary

Introduction

The CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) system is an RNA-based adaptive immune mechanism to degrade invading plasmids and viruses by bacteria and archaea [1]. It was recently shown that a synthetic sgRNA, by fusing crRNA and tracrRNA, can guide Cas endonuclease to target a DNA sequence by design, resulting in site-specific genetic modifications [2,3]. These landmark studies have led to a string of exciting achievements of highly efficient gene targeting in mice and in several other organisms where homologous recombination-based gene targeting strategy was either not available or extremely inefficient [4,5,6,7,8,9,10,11,12]. No success has been reported in employing this system to target the golden Syrian hamster genome

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