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Methods Mol Biol. 2020;2115:445-454. doi: 10.1007/978-1-0716-0290-4_25.

Gene Editing in B-Lymphoma Cell Lines Using CRISPR/Cas9 Technology.

Author information

1
Department of Cancer Immunology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway. baoyan.bai@rr-research.no.
2
KG. Jebsen Centre for B cell Malignancies, University of Oslo, Oslo, Norway. baoyan.bai@rr-research.no.
3
Department of Cancer Immunology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway.
4
KG. Jebsen Centre for B cell Malignancies, University of Oslo, Oslo, Norway.
5
Department of Cellular Therapy, Division of Cancer Medicine, Oslo University Hospital, Oslo, Norway.

Abstract

Genome editing in eukaryotes has greatly improved through the application of targeted editing tools. The development of the CRISPR/Cas9 technology has facilitated genome editing in mammalian cells. However, efficient delivery of CRISPR components into cells growing in suspension remains a challenge. Here, we present a strategy for sequential delivery of the two essential components, Cas9 and sgRNA, into B-lymphoid cell lines. Stable Cas9 expression is obtained by retroviral transduction, before sgRNA is transiently delivered into the Cas9+ cells. This method improves the on-target efficiency of genome editing and, through the transient presence of sgRNA, reduces the potential off-target sites. The current method can be easily applied to other cell types that are difficult to edit with CRISPR/Cas9.

KEYWORDS:

B-lymphoma cells; CRISPR/Cas9; Cas9-expressing cells; Genome editing; Square wave electroporation

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