Generation of B cell-deficient pigs by highly efficient CRISPR/Cas9-mediated gene targeting

J Genet Genomics. 2015 Aug 20;42(8):437-44. doi: 10.1016/j.jgg.2015.05.002. Epub 2015 May 27.

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 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.

Keywords: B cell-deficiency; CRISPR/Cas9 system; IgM heavy chain; Pig genome editing; Somatic cell nuclear transfer.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • B-Lymphocytes / metabolism
  • CRISPR-Cas Systems*
  • Fetus / cytology
  • Fibroblasts
  • Gene Targeting*
  • Genetic Engineering*
  • Immunoglobulin M / genetics*
  • Male
  • Nuclear Transfer Techniques
  • Phenotype
  • Primary Cell Culture
  • Swine

Substances

  • Immunoglobulin M