Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 113, Issue 44, Pages 12514-12519Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1613884113
Keywords
CRISPR/Cas9; sgRNA design; knockout efficiency; primary cells; B cells
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Funding
- European Research Council (ERC) [268921]
- German Ministry of Education and Research within the Validation of the Innovation Potentials of Academic Research (VIP) program [03V0261]
- Berlin Institute of Health Einstein fellowship
- Agence Nationale de la Recherche [ANR-11-BSV3-026-01]
- Institut National du Cancer [13-10/405/AB-LC-HS]
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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.
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