Large insert gene editing in ex vivo, primary human T cells represents an important approach for engineering next-generation cellular immunotherapies. While CRISPR-based editing is well-established for small genetic modifications, large insert knock-in remains a technical bottleneck. This review examines current strategies for achieving efficient, precise and safe integration of large genetic elements. We compare delivery methods for CRISPR components, highlighting electroporation of Cas9 ribonucleoproteins as the most efficient but often cytotoxic approach; emerging alternatives such as cell-penetrating peptides and lipid nanoparticles offer improved cell viability at the expense of editing efficiency. The influence of genomic locus selection, guide RNA design and chromatin context on editing outcomes, as well as the role of homology-directed repair (HDR) template design is evaluated. Various template formats-including linear double- and single-stranded DNA, plasmids, circular single-stranded (css) DNA and recombinant adeno-associated virus (rAAV)-are assessed for their efficiency, toxicity and risk of off-target integration. Where possible, cross-study normalisation is used to enable meaningful comparisons. rAAV templates consistently achieve the highest insertion rates but come with concerns regarding cost, size limitations and insertional mutagenesis, whereas non-viral approaches such as cssDNA templates show promise. Additionally, we review the use of small-molecule modulators to enhance HDR efficiency, noting trade-offs between improved insertion rates and potential genomic instability. Overall, successful large insert editing requires balancing of editing efficiency, cell viability and genomic safety considerations. Continued optimisation of delivery systems, template design and repair pathway modulation will be critical for advancing scalable and clinically viable T-cell therapies.
