Protocol for non-viral HDR-based CRISPR/Cas9 platform for small custom editing in primary T cells

CRISPR/Cas9 enables precision gene editing via homology-directed repair (HDR) for mutation correction and disease modelling. Here we present a customizable T cell single nucleotide variant (SNV) correction platform based on non-viral HDR, previously described in Mamia et al. Precision T Cell Correction Platform for Inborn Errors of Immunity. Molecular Therapy, 2025. To enable reproducible application, we provide a comprehensive, step-by-step 8-day HDR workflow for editing primary T cells, covering CRISPR/Cas9 reagent design, custom genomic editing from start to finish, optimization and validation strategies, on-target detection, and flow cytometric assessment of T cell phenotype and exhaustion. The platform separates locus-specific reagent optimization from a fixed cellular workflow, enabling implementation of new genomic targets without modifying the downstream T cell editing pipeline. Using this workflow, we have achieved up to 80% editing efficiency across multiple loci and donors. We have further optimized the T cell culture platform for PBMC stimulation to promote CD4⁺ and CD8⁺ T cell activation and proliferation. We validated this culture platform in 33 patients with Inborn Errors of Immunity using flow cytometry and observed that the defined culture conditions maintain low T cell exhaustion and support persistence of memory T cell populations. The protocol was developed under research-grade conditions but supports scalability and transition to preclinical and clinical GMP workflows. Overall, this adaptable platform is a broadly applicable framework for precision T cell engineering across diverse genomic targets offering opportunities for both in vitro disease modeling and therapeutic genome correction.