Genome Editing in CAR-T Cells Using CRISPR/Cas9 Technology

CAR-T cell therapy is revolutionizing the treatment of hematologic malignancies. However, there are still many challenges ahead before CAR-T cells can be used effectively to treat solid tumors and certain hematologic cancers, such as T-cell malignancies. Next-generation CAR-T cells containing further genetic modifications are being developed to overcome some of the current limitations of this therapy. In this regard, genome editing is being explored to knock out or knock in genes with the goal of enhancing CAR-T cell efficacy or increasing access. In this chapter, we describe in detail a protocol to knock out genes on CAR-T cells using CRISPR–Cas9 technology. Among various gene editing protocols, due to its simplicity, versatility, and reduced toxicity, we focused on the electroporation of ribonucleoprotein complexes containing the Cas9 protein together with sgRNA. All together, these protocols allow for the design of the knockout strategy, CAR-T cell expansion and genome editing, and analysis of knockout efficiency.