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Journal of Integrative Agriculture  2018, Vol. 17 Issue (2): 406-414    DOI: 10.1016/S2095-3119(17)61748-9
Animal Science · Veterinary Medicine Advanced Online Publication | Current Issue | Archive | Adv Search |
Comparing successful gene knock-in efficiencies of CRISPR/Cas9 with ZFNs and TALENs gene editing systems in bovine and dairy goat fetal fibroblasts
LIU Hui1, 2*, LIU Chang1, 2*, ZHAO Yu-hang1, 2, HAN Xue-jie1, 2, ZHOU Zheng-wei1, 2, WANG Chen1, 2, LI Rong-feng3, 4, LI Xue-ling1, 2
1 State Key Laboratory of Reproductive Regulation & Breeding of Grassland Livestocks, Inner Mongolia University, Hohhot 010070, P.R.China
2 Research Center for Laboratory Animal Science, Inner Mongolia University, Hohhot 010070, P.R.China
3 State Key Laboratories of Reproductive Medicine, Nanjing Medical University, Nanjing 210029, P.R.China
4 Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 210029, P.R.China
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Abstract  This study aimed to compare the efficiencies of clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas9-mediated gene knock-ins with zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) in bovine and dairy goat fetal fibroblasts.  To test the knock-in efficiency, a set of ZFNs and CRISPR/Cas9 plasmids were designed to edit the bovine myostatin (MSTN) gene at exon 2, while a set of TALENs and CRISPR/Cas9 plasmids were designed for editing the dairy goat β-casein gene at exon 2.  Donor plasmids utilizing the ZFNs, TALENs, and CRISPR/Cas9 cutting sites were constructed in the GFP-PGK-NeoR plasmid background, including a 5´ and 3´ homologous arm flanking the genes humanized Fat-1 (hFat-1) or enhanced green fluorescent protein (eGFP).  Subsequently, the ZFNs, TALENs, or CRISPR/Cas9 and the hFat-1 or eGFP plasmids were co-transfected by electroporation into bovine and dairy goat fetal fibroblasts.  After G418 (Geneticin) selection, single cells were obtained by mouth pipetting, flow cytometry or a cell shove.  The gene knock-in events were screened by PCR across the homologous arms.  The results showed that in bovine fetal fibrobalsts, the efficiencies of ZFNs-mediated eGFP and hFat-1 gene knock-ins were 13.68 and 0%, respectively.  The efficiencies of CRISPR/Cas9-mediated eGFP and hFat-1 gene knock-ins were 77.02 and 79.01%, respectively.  The eGFP gene knock-in efficiency using CRISPR/Cas9 was about 5.6 times higher than when using the ZFNs gene editing system.  Additionally, the hFat-1 gene knock-in was only obtained when using the CRISPR/Cas9 system.  The difference of knock-in efficiencies between the ZFNs and CRISPR/Cas9 systems were extremely significant (P<0.01).  In the dairy goat fetal fibroblasts, the efficiencies of TALENs-mediated eGFP and hFat-1 gene knock-ins were 32.35 and 26.47%, respectively.  The efficiencies of eGFP and hFat-1 gene knock-ins using CRISPR/Cas9 were 70.37 and 74.29%, respectively.  The knock-in efficiencies difference between the TALENs and CRISPR/Cas9 systems were extremely significant (P<0.01).  This study demonstrated that CRISPR/Cas9 was more efficient at gene knock-ins in domesticated animal cells than ZFNs and TALENs.  The CRISPR/Cas9 technology offers a new era of precise gene editing in domesticated animal cell lines. 
Keywords:  myostatin (MSTN)        β-casein (CSN2)        bovine fetal fibroblasts        CRISPR/Cas9        dairy goat fetal fibroblasts        eGFP        hFat-1        knock-in        mutation efficiency       TALENs        ZFNs  
Received: 13 March 2017   Accepted:

This study was supported by the National Transgenic Project of China (2016ZX08010001-002), the National Natural Science Foundation of China (81471001), the Inner Mongolia Science and Technology Program, China (201502073), and the National 863 Prgram of China (2009AA10Z111).

Corresponding Authors:  Correspondence LI Xue-ling, E-mail:    
About author:  LIU Hui, E-mail:; LIU Chang, E-mail:; * These authors contributed equally to this study.

Cite this article: 

LIU Hui, LIU Chang, ZHAO Yu-hang, HAN Xue-jie, ZHOU Zheng-wei, WANG Chen, LI Rong-feng, LI Xue-ling . 2018. Comparing successful gene knock-in efficiencies of CRISPR/Cas9 with ZFNs and TALENs gene editing systems in bovine and dairy goat fetal fibroblasts. Journal of Integrative Agriculture, 17(2): 406-414.

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