An efficient and cost-effective method for disrupting genes in RAW264.7 macrophages using CRISPR-Cas9.

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) are widely used for genome editing in cultured cell lines. However, the implementation of genome editing is still challenging due to the complex and often costly multi-step process associated with this technique. Moreover, the efficiency of genome editing varies across cell types, often limiting utility. Herein, we describe pCRISPR-EASY, a vector for simplified cloning of single guide RNAs (sgRNAs) and its simultaneous introduction with CRISPR-Cas9 into cultured cells using a non-viral delivery system. We outline a comprehensive, step-by-step protocol for genome editing in RAW264.7 macrophages, a mouse macrophage cell line widely used in biomedical research for which genome editing using CRISPR-Cas9 has been restricted to lentiviral or expensive commercial reagents. This provides an economical, highly efficient and reliable method for genome editing that can easily be adapted for use in other systems.