Optimizing CRISPR–Cas9-mediated TCR knockout in primary mouse CD8+, CD4+, and regulatory T cells

CRISPR–Cas9 genome editing has transformed biomedical research by enabling precise and efficient genetic modification. Among its applications, gene knockout using ribonucleoprotein (RNP) complexes provides a non-viral strategy for introducing loss-of-function mutations. Here, we present an optimized CRISPR–Cas9 RNP-based genome editing pipeline for T cells, beginning with human Jurkat cells and extending to primary mouse CD4⁺, CD8⁺, and regulatory T cells (Tregs). We benchmarked the workflow by targeting the T cell receptor alpha constant ( TRAC/Trac ) locus, an important site for engineering therapeutic T cells. In Jurkat cells, optimized RNP conditions yielded > 98% knockout efficiency. In primary mouse T cells, we achieved up to 95% and 80% TCR knockout in CD8⁺ and CD4⁺ subsets, respectively, with minimal effects on viability. CRISPR–Cas9-mediated editing was validated by Sanger sequencing and Inference of CRISPR Edits (ICE) analysis, confirming efficient on-target indel formation consistent with loss of surface CD3 expression. Edited cells retained their capacity for activation. The sgRNA design, the Cas9:sgRNA molar ratio, cell number, and RNP dose were key determinants of editing efficiency. Notably, we demonstrate efficient Trac editing in primary mouse Tregs, reaching up to 85% knockout. Although Tregs displayed modestly reduced viability after electroporation, editing remained robust. Overall, this work provides an optimized non‑viral genome editing platform for mouse T cells, supporting mechanistic studies in syngeneic models and the development of next‑generation cellular immunotherapies.