Generation of B cell-deficient pigs by highly efficient CRISPR/Cas9-mediated gene targeting.
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 JH 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.
State Key Laboratory of Reproductive Medicine and Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 210029, China.
Fengjiao
F
Chen
State Key Laboratory of Reproductive Medicine and Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 210029, China.
Ying
Y
Wang
State Key Laboratory of Reproductive Medicine and Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 210029, China.
Yilin
Y
Yuan
State Key Laboratory of Reproductive Medicine and Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 210029, China.
Wei
W
Zhang
State Key Laboratory of Reproductive Medicine and Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 210029, China.
Zijian
Z
Ren
State Key Laboratory of Reproductive Medicine and Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 210029, China.
Yong
Y
Jin
State Key Laboratory of Reproductive Medicine and Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 210029, China.
Xiaorui
X
Liu
State Key Laboratory of Reproductive Medicine and Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 210029, China.
Qiang
Q
Xiong
State Key Laboratory of Reproductive Medicine and Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 210029, China.
Qin
Q
Chen
State Key Laboratory of Reproductive Medicine and Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 210029, China.
Manling
M
Zhang
State Key Laboratory of Reproductive Medicine and Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 210029, China.
Xiaokang
X
Li
State Key Laboratory of Reproductive Medicine and Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 210029, China.
Lihua
L
Zhao
State Key Laboratory of Reproductive Medicine and Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 210029, China.
Ze
Z
Li
State Key Laboratory of Reproductive Medicine and Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 210029, China.
Zhaoqiang
Z
Wu
State Key Laboratory of Reproductive Medicine and Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 210029, China.
Yanfei
Y
Zhang
State Key Laboratory of Reproductive Medicine and Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 210029, China.
Feifei
F
Hu
State Key Laboratory of Reproductive Medicine and Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 210029, China.
Juan
J
Huang
State Key Laboratory of Reproductive Medicine and Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 210029, China. Electronic address: lirongfeng@njmu.edu.cn.
Rongfeng
R
Li
State Key Laboratory of Reproductive Medicine and Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 210029, China. Electronic address: daiyifan@njmu.edu.cn.
Yifan
Y
Dai
D000818:Animals; D030801:Animals, Genetically Modified; D001402:B-Lymphocytes / Q000378:metabolism; D064113:CRISPR-Cas Systems; D005333:Fetus / Q000166:cytology; D005347:Fibroblasts; D018390:Gene Targeting; D005818:Genetic Engineering; D007075:Immunoglobulin M / Q000235:genetics*; D008297:Male; D053652:Nuclear Transfer Techniques; D010641:Phenotype; D061251:Primary Cell Culture; D013552:Swine
D007075:Immunoglobulin M
B cell-deficiency; CRISPR/Cas9 system; IgM heavy chain; Pig genome editing; Somatic cell nuclear transfer