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.