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| Item Type: | Article |
|---|---|
| Title: | Efficient CRISPR-mediated mutagenesis in primary immune cells using CrispRGold and a C57BL/6 Cas9 transgenic mouse line |
| Creators Name: | Chu, V.T., Graf, R., Wirtz, T., Weber, T., Favret, J., Li, X., Petsch, K., Tran, N.T., Sieweke, M.H., Berek, C., Kühn, R. and Rajewsky, K. |
| 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: | CRISPR/Cas9, sgRNA Design, Knockout Efficiency, Primary Cells, B Cells, Animals, Mice |
| Source: | Proceedings of the National Academy of Sciences of the United States of America |
| ISSN: | 0027-8424 |
| Publisher: | National Academy of Sciences |
| Volume: | 113 |
| Number: | 44 |
| Page Range: | 12514-12519 |
| Date: | 1 November 2016 |
| Official Publication: | https://doi.org/10.1073/pnas.1613884113 |
| PubMed: | View item in PubMed |
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