There are numerous mechanisms enabling a cell to silence a transgene, including active epigenetic silencing of the gene or transcript through recognition of viral cis elements 17, 18, 19. Semi-random integration of genetic payloads into primary cells using lentiviruses or retroviruses often leads to a reduction (or even complete loss) of transgene expression over time 16. Transgene silencing is a major hindrance to using these tools for sophisticated cell engineering and therapy 14, 15. Biomolecular tools such as next-generation receptors 4, 5, 6, clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) and CRISPR interference (CRISPRi) 7, 8, 9, 10, and logic gates 11, 12, 13 could enhance this therapeutic modality, but their long-term expression often poses a major challenge in primary cells. Efforts to improve their functionality, reduce toxicity or move beyond blood cancer into solid tumours, ageing, autoimmunity and viral clearance have leveraged combinations of tools that increase the ability of T cells to sense, process and respond to the disease. For example, chimeric antigen receptor (CAR) T cells leverage a single engineered receptor to rewire its cytotoxicity towards cancers 2, 3. Maximizing the therapeutic potential and improving the efficacy and safety of engineered cells often require the expression of large genes or of complex gene circuits 1. CLIP offers a scalable and efficient method for manufacturing engineered primary cells. We show that CLIP enables the efficient insertion and stable expression of large payloads and of two difficult-to-express viral antigens in primary T cells at low cytotoxicity. The method, which we named CLIP (for ‘CRISPR for long-fragment integration via pseudovirus’), leverages an integrase-deficient lentivirus encoding a payload flanked by homology arms and ‘cut sites’ to insert the payload upstream and in-frame of an endogenous essential gene, followed by the delivery of a CRISPR-associated ribonucleoprotein complex via electroporation. Here we report a method for the knock-in and stable expression of a large payload and for the simultaneous knock-in of two genes at two endogenous loci. Leveraging homology-directed repair to place payloads under the control of endogenous essential genes can overcome silencing but often results in low knock-in efficiencies and cytotoxicity. Large payload insertion via retroviruses is typically semi-random and hindered by transgene silencing. The targeted insertion and stable expression of a large genetic payload in primary human cells demands methods that are robust, efficient and easy to implement.
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