Targeted Gene Editing in Human Primary T Cells Using CRISPR/Cas9 Ribonucleoproteins

Background: Clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9 (CRISPR/Cas9)-mediated gene knockout of primary T cell has several limitations for clinical applications. Direct delivery of recombinant Cas9 protein and synthetic gRNA, as a pre-assembled ribonucleoprotein (RNP) complex, has become a potent approach to introduce highly efficient gene editing in primary T cells. In this study, we employed Cas9 RNP-based delivery system for targeted T Cell receptor alpha constant (TRAC) and β2 microglobulin (B2M) genes knockout in human primary T cells. Methods: Specific gRNAs were designed to target the first exons of TRAC and B2M genes. Cas9 protein and respective synthetic gRNAs were then mixed separately, and electroporated into human primary T cells isolated from peripheral blood mononuclear cells (PBMCs). The gene editing efficiency was quantified using tracking of indels by decomposition (TIDE) analysis and flow cytometry. Findings: Three days after electroporation of primary T cells with the TRAC and B2M targeting RNP complexes, TIDE analysis revealed the knockout efficiency of 13-60 percent for the TRAC-targeting gRNAs and 21-53 percent for B2M-targeting gRNAs. Flow cytometry analysis confirmed ~76% and ~27% complete loss of expression for the most efficient gRNAs targeting TRAC (TRAC-gRNA3) and B2M (B2M-gRNA2), respectively. Conclusion: Our results demonstrate that Cas9 RNP system can be efficiently delivered into primary T cells and result in targeted gene knockout. The protocol described here enables a streamlined and highly efficient solution for maximizing editing efficiency in primary T cells, and simplifies the gene editing process for next-generation immunotherapies.