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

Abstract

CRISPR/Cas9 enables precision gene editing via HDR for mutation correction and disease modelling. This protocol describes an 8-day non-viral HDR workflow for editing primary patient and healthy donor T cells, including reagent design, editing, on-target detection, and flow cytometry. The protocol was developed under research-grade conditions but supports scaling up and the transition to preclinical and clinical GMP workflows. For complete details on the use and execution of this protocol, please refer to Mamia et al.[1]. Before you beginCRISPR/Cas9 gene editing is a promising tool to correct pathogenic variants for autologous cell therapies, targeting monogenic diseases such as inborn errors of immunity (IEI). Furthermore, it can be used as a tool for disease modelling to study normal and pathological variations of the immune system. Here we present a detailed protocol for an efficient and customizable T cell single nucleotide variant (SNV) correction platform based on homology-directed repair (HDR). The protocol details every step of the process, which starts with custom CRISPR/Cas9 reagent design of guide-RNAs (gRNAs) and repair templates for editing a novel target with no previously published reagents. Furthermore, we describe the strategy of reagent design to assess on-target HDR editing using droplet digital PCR (ddPCR). Next, we detail the T cell platform itself, and present effective strategies to stimulate PBMCs ex vivo to promote CD4+ and CD8+ T cell activation and proliferation, which we have validated in 32 unique IEI patients. Next, we present the workflow of gene editing T cells using nucleofection and CRISPR ribonucleoprotein (RNP) complexes for efficient editing that preserves high cell viabilities and up to 80% HDR. Finally, we present a flow cytometry panel that assesses the immune cells present at the end of the platform, including characterization of memory and effector T cell populations and status of T cell exhaustion. InnovationIn the study, we present a detailed protocol of performing highly efficient, non-viral and HDR-based precision editing in patient and healthy control T cells. The developed platform enables custom editing, such as correction of small pathogenic variants, with one workflow that we demonstrate to achieve up to 80% efficiency in multiple genomic loci and donors. Institutional permissionsThe study was conducted in accordance with the principles of the Helsinki Declaration and approved by the Helsinki University Central Hospital Ethics Committee, and the Regional Committee for Medical and Health Research Ethics South-East Norway. All participants have signed written informed consent.