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

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.

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.

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.

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