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Cellular and Molecular Life Sciences

, Volume 72, Issue 6, pp 1175–1184 | Cite as

Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer

  • Xiaoqing Zhou
  • Jige Xin
  • Nana Fan
  • Qingjian Zou
  • Jiao Huang
  • Zhen Ouyang
  • Yu Zhao
  • Bentian Zhao
  • Zhaoming Liu
  • Sisi Lai
  • Xiaoling Yi
  • Lin Guo
  • Miguel A. Esteban
  • Yangzhi Zeng
  • Huaqiang Yang
  • Liangxue Lai
Research Article

Abstract

The domestic pig has been widely used as an important large animal model. Precise and efficient genetic modification in pig provides a great promise in biomedical research. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has been successfully used to produce many gene-targeted animals. However, these animals have been generated by co-injection of Cas9 mRNA and single-guide RNA (sgRNA) into one-cell stage embryos, which mostly resulted in mosaicism of the modification. One or two rounds of further breeding should be performed to obtain homozygotes with identical genotype and phenotype. To address this issue, gene-targeted somatic cells can be used as donor for somatic cell nuclear transfer (SCNT) to produce gene-targeted animals with single and identical mutations. In this study, we applied Cas9/sgRNAs to effectively direct gene editing in porcine fetal fibroblasts and then mutant cell colonies were used as donor to generate homozygous gene-targeted pigs through single round of SCNT. As a result, we successfully obtained 15 tyrosinase (TYR) biallelic mutant pigs and 20 PARK2 and PINK1 double-gene knockout (KO) pigs. They were all homozygous and no off-target mutagenesis was detected by comprehensive analysis. TYR −/− pigs showed typical albinism and the expression of parkin and PINK1 were depleted in PARK2 −/−/PINK1 −/− pigs. The results demonstrated that single- or double-gene targeted pigs can be effectively achieved by using the CRISPR/Cas9 system combined with SCNT without mosaic mutation and detectable off-target effects. This gene-editing system provides an efficient, rapid, and less costly manner to generate genetically modified pigs or other large animals.

Keywords

Albinism Animal model Genome engineering Parkinson’s disease 

Abbreviations

Cas

CRISPR-associated nuclease

CRISPR

Clustered regularly interspaced short palindromic repeats

KO

Knockout

OTS

Off-target site

PFF

Porcine fetal fibroblast

PINK1

PTEN-induced putative kinase 1

SCNT

Somatic cell nuclear transfer

sgRNA

Single-guide RNA

TYR

Tyrosinase

WT

Wild type

Notes

Acknowledgments

This study was supported by the Natural Science Foundation of China (Nos. 31301103 and 31360532) to H.Y. and J.X., the National Basic Research Program of China (973 program; No. 2011CB944203) to L. L.

Conflict of interest

The authors have declared no conflict of interest.

Supplementary material

18_2014_1744_MOESM1_ESM.doc (154 kb)
Supplementary material 1 (DOC 154 kb)
18_2014_1744_MOESM2_ESM.doc (356 kb)
Supplementary material 2 (DOC 356 kb)

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Copyright information

© Springer Basel 2014

Authors and Affiliations

  • Xiaoqing Zhou
    • 1
  • Jige Xin
    • 2
    • 3
  • Nana Fan
    • 1
  • Qingjian Zou
    • 1
  • Jiao Huang
    • 1
  • Zhen Ouyang
    • 1
  • Yu Zhao
    • 1
  • Bentian Zhao
    • 1
  • Zhaoming Liu
    • 1
  • Sisi Lai
    • 1
  • Xiaoling Yi
    • 1
  • Lin Guo
    • 1
  • Miguel A. Esteban
    • 1
  • Yangzhi Zeng
    • 3
  • Huaqiang Yang
    • 1
  • Liangxue Lai
    • 1
    • 2
  1. 1.South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and HealthChinese Academy of SciencesGuangzhouChina
  2. 2.Jilin Provincial Key Laboratory of Animal Embryo Engineering, Institute of Zoonosis, College of Veterinary MedicineJilin UniversityChangchunChina
  3. 3.Key Laboratory of Banna Mini-pig Inbred Line of Yunnan Province, Animal Science and Technology CollegeYunnan Agricultural UniversityKunmingChina

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