MINI REVIEW article
Front. Immunol.
Sec. Cancer Immunity and Immunotherapy
This article is part of the Research TopicSafety and efficacy of CRISPR/Cas-based genome editing tools: applications and considerations in cell and gene therapyView all articles
Engineering with Care: Safety Assessment Platforms for CRISPR-Modified Natural Killer cells
Provisionally accepted
- 1Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, Freiburg, Germany
- 2PhD Program, Faculty of Biology, University of Freiburg, Freiburg, Germany
- 3Goethe University Frankfurt, Department of Pediatrics, Frankfurt am Main, Germany
- 4Goethe University Frankfurt, Frankfurt Cancer Institute, Frankfurt am Main, Germany
- 5German Cancer Consortium (DKTK) partner site Frankfurt/Mainz, Frankfurt am Main, Germany
- 6Center for Cell and Gene Therapy, Medical Center - University of Freiburg, Freiburg, Germany
- 7German Cancer Consortium (DKTK) partner site Freiburg, Freiburg, Germany
- 8Faculty of Medicine, University of Freiburg, Freiburg, Germany
- 9Department of Pediatrics, Goethe University Frankfurt, Frankfurt, Germany
- 10German Red Cross Blood Service Baden-Württemberg - Hessen, Institute for Transfusion Medicine and Immunohematology, Frankfurt am Main, Germany
- 11Berlin Center for Advanced Therapies, Charité – Universitätsmedizin Berlin, Berlin, Germany
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CRISPR-based gene editing has become a transformative tool to enhance immune cell therapies. In particular, engineering natural killer (NK) cells with CRISPR/Cas systems has gained traction due to their ability to mediate strong anti-tumor responses in an MHC-unrestricted, non-alloreactive manner. Early trials show the feasibility and safety of allogeneic NK cells, paving the way as scalable "off-the-shelf" products. CRISPR/Cas9 edits genomes by inducing DNA double-strand breaks (DSBs), mainly repaired through non-homologous end joining (NHEJ) or homology-directed repair (HDR). While effective, CRISPR carries risks of off-target (OT) activity that may disrupt essential genes, cause chromosomal rearrangements, or trigger oncogenic changes - posing threats to product integrity and patient safety. These concerns intensify with multiplex editing, where multiple loci are modified to improve function, persistence, and immune evasion. Since unmodified NK cells are typically short-lived, many clinical-stage products are engineered to express IL-15 or related constructs, extending their half-life and amplifying risks associated with unintended changes. This underscores the urgent need for robust safety assessments. In this review, we summarize the current landscape of safety assessment platforms for evaluating gene edited NK cells. We highlight predictive in silico tools, biochemical in vitro assays, and emerging cell-based detection systems to identify and quantify CRISPR-induced OT events. Particular attention is given to their suitability, limitations, and practical use in primary NK cells and multiplex editing strategies. Our aim is to support the design of safe, effective editing workflows for NK cell therapies - ensuring rigor as the field advances rapidly toward clinical application.
Keywords: CRISPR, NK cells, Safty assessment, off-target, Allogenic, Gene-editing, cell therapy
Received: 23 Sep 2025; Accepted: 10 Nov 2025.
Copyright: © 2025 Fazeli, Ullrich, Cathomen and Bexte. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Tobias Bexte, bexte@med.uni-frankfurt.de
Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.