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CRISPR/Cas9-mediated in vitro mutagenesis in GC-like B cells

Authors

  • V.T. Chu
  • R. Graf
  • K. Rajewsky

Journal

  • Methods in Molecular Biology

Citation

  • Methods Mol Biol 1623: 135-145

Abstract

  • The CRISPR/Cas9 technology has developed into a powerful tool for genome editing, both in terms of gene silencing and the insertion of precise mutations. However, the application of CRISPR/Cas9-mediated mutagenesis in primary immune cells, in particular in B cells, is still in its infancy because of the difficulty to deliver the CRISPR/Cas9 system into these cells. Here, we describe a new method to use CRISPR/Cas9 for manipulating genes in germinal center (GC)-like B cells in vitro. We isolated Cas9-expressing B cells from R26-Cas9iGFP/+ mice (expressing Cas9 constitutively from the Rosa26 locus) and mixed them with control B cells. Primary B cells were cultured on CD40L- and BAFF-expressing feeder cells and transduced with retroviral particles expressing the sgRNAs of interest. Using this system, we have achieved complete gene knockouts in up to 92% of activated B cells.


DOI

doi:10.1007/978-1-4939-7095-7_12