Efficient CRISPR-mediated mutagenesis in primary immune cells using CrispRGold and a C57BL/6 Cas9 transgenic mouse line


  • V.T. Chu
  • R. Graf
  • T. Wirtz
  • T. Weber
  • J. Favret
  • X. Li
  • K. Petsch
  • N.T. Tran
  • M.H. Sieweke
  • C. Berek
  • R. Kühn
  • K. Rajewsky


  • Proceedings of the National Academy of Sciences of the United States of America


  • Proc Natl Acad Sci U S A 113 (44): 12514-12519


  • Applying clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-mediated mutagenesis to primary mouse immune cells, we used high-fidelity single guide RNAs (sgRNAs) designed with an sgRNA design tool (CrispRGold) to target genes in primary B cells, T cells, and macrophages isolated from a Cas9 transgenic mouse line. Using this system, we achieved an average knockout efficiency of 80% in B cells. On this basis, we established a robust small-scale CRISPR-mediated screen in these cells and identified genes essential for B-cell activation and plasma cell differentiation. This screening system does not require deep sequencing and may serve as a precedent for the application of CRISPR/Cas9 to primary mouse cells.