Efficient CRISPR-mediated mutagenesis in primary immune cells using CrispRGold and a C57BL/6 Cas9 transgenic mouse line.
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.
['Chu|Van Trung|VT|', 'Graf|Robin|R|', 'Wirtz|Tristan|T|', 'Weber|Timm|T|', 'Favret|Jeremy|J|', 'Li|Xun|X|', 'Petsch|Kerstin|K|', 'Tran|Ngoc Tung|NT|', 'Sieweke|Michael H|MH|', 'Berek|Claudia|C|', 'Kühn|Ralf|R|', 'Rajewsky|Klaus|K|']
D000818:Animals D001402:B-Lymphocytes D064113:CRISPR-Cas Systems D002454:Cell Differentiation D002478:Cells, Cultured D000072669:Gene Editing D008213:Lymphocyte Activation D008264:Macrophages D008807:Mice, Inbred BALB C D008810:Mice, Inbred C57BL D008822:Mice, Transgenic D016296:Mutagenesis D010950:Plasma Cells D015203:Reproducibility of Results D013601:T-Lymphocytes
sgRNA design B cells knockout efficiency CRISPR/Cas9 primary cells