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

Item Type:Article
Title:CRISPR/Cas9-mediated in vitro mutagenesis in GC-like B cells
Creators Name:Chu, V.T. and Graf, R. and Rajewsky, K.
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.
Keywords:B Cells, 40LB Feeder Cell, CRISPR/Cas9, Genome Editing, In Vitro, Animals, Mice
Source:Methods in Molecular Biology
Series Name:Methods in Molecular Biology
Title of Book:Germinal Centers : Methods and Protocols
ISSN:1064-3745
ISBN:978-1-4939-7094-0
Publisher:Springer / Humana Press (U.S.A.)
Volume:1623
Page Range:135-145
Date:2017
Official Publication:https://doi.org/10.1007/978-1-4939-7095-7_12
PubMed:View item in PubMed

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