Generation of B Cell-Deficient Pigs by Highly Efficient CRISPR/Cas9-Mediated Gene Targeting

Fengjiao Chen; Ying Wang; Yilin Yuan; Wei Zhang; Zijian Ren; Yong Jin; Xiaorui Liu; Qiang Xiong; Qin Chen; Manling Zhang; Xiaokang Li; Lihua Zhao; Ze Li; Zhaoqiang Wu; Yanfei Zhang; Feifei Hu; Juan Huang; Rongfeng Li; Yifan Dai

doi:10.1016/j.jgg.2015.05.002

Volume 42 Issue 8

Aug. 2015

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Generation of B Cell-Deficient Pigs by Highly Efficient CRISPR/Cas9-Mediated Gene Targeting

doi: 10.1016/j.jgg.2015.05.002

State Key Laboratory of Reproductive Medicine and Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 210029, China

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Corresponding author: E-mail address: lirongfeng@njmu.edu.cn (Rongfeng Li); E-mail address: daiyifan@njmu.edu.cn (Yifan Dai)
Received Date: 2015-02-09
Accepted Date: 2015-05-19
Rev Recd Date: 2015-05-01

Available Online: 2015-05-27

Publish Date: 2015-08-20

Abstract

Abstract

Generating B cell-deficient mutant is the first step to produce human antibody repertoires in large animal models. In this study, we applied the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system to target the J_H region of the pig IgM heavy chain gene which is crucial for B cell development and differentiation. Transfection of IgM-targeting Cas9 plasmid in primary porcine fetal fibroblasts (PFFs) enabled inducing gene knock out (KO) in up to 53.3% of colonies analyzed, a quarter of which harbored biallelic modification, which was much higher than that of the traditional homologous recombination (HR). With the aid of somatic cell nuclear transfer (SCNT) technology, three piglets with the biallelic IgM heavy chain gene mutation were produced. The piglets showed no antibody-producing B cells which indicated that the biallelic mutation of the IgM heavy chain gene effectively knocked out the function of the IgM and resulted in a B cell-deficient phenotype. Our study suggests that the CRISPR/Cas9 system combined with SCNT technology is an efficient genome-editing approach in pigs.
- CRISPR/Cas9 system,
- Pig genome editing,
- B cell-deficiency,
- Somatic cell nuclear transfer

These authors contributed equally to this work.

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References(38)

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