Comparing successful gene knock-in efficiencies of CRISPR/Cas9 with ZFNs and TALENs gene editing systems in bovine and dairy goat fetal fibroblasts

doi:10.1016/S2095-3119(17)61748-9

Journal of Integrative Agriculture ›› 2018, Vol. 17 ›› Issue (2): 406-414.DOI: 10.1016/S2095-3119(17)61748-9

收稿日期:2017-03-13 出版日期:2018-02-20 发布日期:2018-02-01

Comparing successful gene knock-in efficiencies of CRISPR/Cas9 with ZFNs and TALENs gene editing systems in bovine and dairy goat fetal fibroblasts

LIU Hui^{1, 2*}, LIU Chang^{1, 2*}, ZHAO Yu-hang^{1, 2}, HAN Xue-jie^{1, 2}, ZHOU Zheng-wei^{1, 2}, WANG Chen^{1, 2}, LI Rong-feng^{3, 4}, LI Xue-ling^{1, 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

Received:2017-03-13 Online:2018-02-20 Published:2018-02-01
Contact: Correspondence LI Xue-ling, E-mail: lixueling@hotmail.com
About author:LIU Hui, E-mail: liuhuicaoyuan@foxmail.com; LIU Chang, E-mail: 1551564908@qq.com; * These authors contributed equally to this study.
Supported by:
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).

摘要/Abstract

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.

Key words: myostatin (MSTN) , β-casein (CSN2) , bovine fetal fibroblasts , CRISPR/Cas9 , dairy goat fetal fibroblasts , eGFP , hFat-1 , knock-in , mutation efficiency , TALENs , ZFNs

. [J]. Journal of Integrative Agriculture, 2018, 17(2): 406-414.

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

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Comparing successful gene knock-in efficiencies of CRISPR/Cas9 with ZFNs and TALENs gene editing systems in bovine and dairy goat fetal fibroblasts

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