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Peer-reviewedCC BY 4.0Frontiers in Genome Editing

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

  • Katariina Mamia1,2,3
  • Anniken Solveig Matheson Sollano1,3,4
  • Vanja Lundberg1,5,6
  • Svetlana Vakkilainen7
  • Eliisa Kekäläinen8,9,10
  • Camilla Schalin-Jäntti11,12
  • Eira Leinonen13
  • Børre Fevang14,15
  • Juha Grönholm9,16,17
  • Timi Martelius18
  • Shiva Dahal-Koirala1,3
  • Emma Maria Haapaniemi1,2,3
18 affiliations
  1. 1Norwegian Centre for Molecular Biosciences and Medicine, University of Oslo, Oslo, Norway
  2. 2Department of Pediatrics, Oslo University Hospital, Oslo, Norway
  3. 3Precision Immunotherapy Alliance, University of Oslo, Oslo, Norway
  4. 4School of Biosciences and Medicine, University of Surrey, Guildford, Surrey, United Kingdom
  5. 5Department of Pediatrics, Institute of Clinical Sciences, the Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden
  6. 6Department of Rheumatology and Inflammation Research, Institute of Medicine, the Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden
  7. 7Children’s Hospital and Pediatric Research Center, University of Helsinki and Helsinki University Hospital, Helsinki, Finland
  8. 8Clinical Microbiology, HUS Diagnostic Center, Helsinki University Hospital and University of Helsinki, Helsinki, Finland
  9. 9Translational Immunology Research Program, University of Helsinki, Helsinki, Finland
  10. 10Institute for Molecular Medicine Finland and Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland
  11. 11Endocrinology, Abdominal Center, Helsinki University Hospital, Helsinki, Finland
  12. 12University of Helsinki, ENDO-ERN (European Reference Network on Rare Endocrine Conditions), Helsinki, Finland
  13. 13Folkhälsan Institute of Genetics, and Stem Cells and Metabolism Research Program, University of Helsinki, Helsinki, Finland
  14. 14Institute of Clinical Medicine, University of Oslo, Oslo, Norway
  15. 15Section of Clinical Immunology and Infectious Diseases, Oslo University Hospital, Rikshospitalet, Oslo, Norway
  16. 16Pediatric Research Center, New Children’s Hospital, University of Helsinki and HUS Helsinki University Hospital, Helsinki, Finland
  17. 17Division of Hematology, Oncology, and Stem Cell Transplantation, New Children’s Hospital, HUS Helsinki University Hospital, Helsinki, Finland
  18. 18Inflammation Center, Department of Infectious Disease, Helsinki University Hospital and University of Helsinki, Helsinki, Finland
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Abstract

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.

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Cite

BibTeXRIS
BibTeX
@article{mamia2026protocol,
  title = {{Protocol for non-viral HDR-based CRISPR/Cas9 platform for small custom editing in primary T cells}},
  author = {Katariina Mamia and Anniken Solveig Matheson Sollano and Vanja Lundberg and Svetlana Vakkilainen and Eliisa Kekäläinen and Camilla Schalin-Jäntti and Eira Leinonen and Børre Fevang and Juha Grönholm and Timi Martelius and Shiva Dahal-Koirala and Emma Maria Haapaniemi},
  journal = {Frontiers in Genome Editing},
  year = {2026},
  month = sep,
  doi = {10.3389/fgeed.2026.1817322},
  url = {https://doi.org/10.3389/fgeed.2026.1817322},
  note = {CC BY 4.0},
}
RIS
TY  - JOUR
TI  - Protocol for non-viral HDR-based CRISPR/Cas9 platform for small custom editing in primary T cells
AU  - Katariina Mamia
AU  - Anniken Solveig Matheson Sollano
AU  - Vanja Lundberg
AU  - Svetlana Vakkilainen
AU  - Eliisa Kekäläinen
AU  - Camilla Schalin-Jäntti
AU  - Eira Leinonen
AU  - Børre Fevang
AU  - Juha Grönholm
AU  - Timi Martelius
AU  - Shiva Dahal-Koirala
AU  - Emma Maria Haapaniemi
PY  - 2026
DA  - 2026/09/30
JO  - Frontiers in Genome Editing
DO  - 10.3389/fgeed.2026.1817322
UR  - https://doi.org/10.3389/fgeed.2026.1817322
AB  - 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.
SN  - 2673-3439
ER  -