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

Proc Natl Acad Sci U S A. 2016 Nov 1;113(44):12514-12519. doi: 10.1073/pnas.1613884113. Epub 2016 Oct 11.

Abstract

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.

Keywords: B cells; CRISPR/Cas9; knockout efficiency; primary cells; sgRNA design.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • B-Lymphocytes / metabolism*
  • CRISPR-Cas Systems*
  • Cell Differentiation / genetics
  • Cells, Cultured
  • Gene Editing / methods*
  • Lymphocyte Activation / genetics
  • Macrophages / metabolism*
  • Mice, Inbred BALB C
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Mutagenesis*
  • Plasma Cells / metabolism
  • Reproducibility of Results
  • T-Lymphocytes / metabolism*