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

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

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.

MeSH terms

  • B-Lymphocytes / metabolism
  • CRISPR-Associated Protein 9 / genetics
  • CRISPR-Cas Systems*
  • Cell Line, Tumor
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Electroporation / methods
  • Gene Editing / methods*
  • Gene Transfer Techniques
  • Humans
  • Lymphoma, B-Cell / genetics*
  • Lymphoma, B-Cell / therapy
  • RNA, Guide / genetics
  • Transduction, Genetic / methods

Substances

  • RNA, Guide
  • CRISPR-Associated Protein 9