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Journal of Integrative Agriculture  2019, Vol. 18 Issue (6): 1329-1336    DOI: 10.1016/S2095-3119(19)62694-8
Animal Science · Veterinary Medicine Advanced Online Publication | Current Issue | Archive | Adv Search |
Generation of pigs with a Belgian Blue mutation in MSTN using CRISPR/ Cpf1-assisted ssODN-mediated homologous recombination
ZOU Yun-long1, 2, LI Zhi-yuan2, ZOU Yun-jing3, HAO Hai-yang2, HU Jia-xiang2, LI Ning2, LI Qiu-yan2  
1 State Key Laboratory of Silkworm Genome Biology, Key Laboratory of Sericultural Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs, College of Biotechnology, Southwest University, Chongqing 400715, P.R.China
2 State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, P.R.China
3 College of Veterinary Medicine, China Agricultural University, Beijing 100193, P.R.China 
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CRISPR/Cpf1 has emerged recently as an effective tool for genome editing in many organisms, but its use in pigs to generate precise genetic modifications has seldom been described.  Myostatin (MSTN) is a well-characterized negative regulator of muscle development, and natural mutations in this gene cause a double-muscled phenotype in many species.  However, to the best of our knowledge, no naturally occurring mutation in MSTN has been found in pigs.  In addition, no living pig models with sophisticated modifications orthologous to natural mutations in MSTN have yet been reported.  In this study, we exploited the CRISPR/Cpf1 system to introduce a predefined modification orthologous to the natural MSTN mutation found in Belgian Blue cattle (thus known as the Belgian Blue mutation).  Our research demonstrated that the cutting efficiency of CRISPR/Cpf1 was 12.3% in mixed porcine fetal fibroblasts in drug free medium, and 41.7% in clonal colonies obtained using G418 selection.  Then, the Cpf1-sgRNA vector, ssODN template, and a self-excision cassette were co-transfected into porcine fetal fibroblasts.  After G418 selection, 8 clonal colonies were examined and 5 with genetic modification were found.  Of these 5, 2 harbored the precise 11-bp deletion.  Using 1 heterozygous clonal colony, 2 cloned Duroc piglets were successfully generated, which was heterozygous for the Belgian Blue mutation.  In summary, our results demonstrate that CRISPR/Cpf1 system can be used efficiently to generate double-stranded breaks, and also to mediate homologous recombination to introduce precise genomic modifications in pigs.
Keywords:  MSTN        CRISPR/Cpf1        Belgian Blue mutation        genetically modified pigs        single stranded oligodeoxynucleotide  
Received: 03 September 2018   Accepted:
Fund: This work was supported by the National Transgenic Breeding Program of China (2016ZX08006001) and the Doctor’s Fund of Southwest University, China (SWU 118082).
Corresponding Authors:  Correspondence LI Qiu-yan, Tel: +86-10-62732334, E-mail:    
About author:  ZOU Yun-long, Tel: +86-23-68251309, E-mail:;

Cite this article: 

ZOU Yun-long, LI Zhi-yuan, ZOU Yun-jing, HAO Hai-yang, HU Jia-xiang, LI Ning, LI Qiu-yan. 2019. Generation of pigs with a Belgian Blue mutation in MSTN using CRISPR/ Cpf1-assisted ssODN-mediated homologous recombination. Journal of Integrative Agriculture, 18(6): 1329-1336.

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