ONE-STEP tagging: a versatile method for rapid site-specific integration by simultaneous reagent delivery
Summary
We present a novel, versatile genome editing method termed ONE-STEP tagging, which combines CRISPR-Cas9-mediated targeting with Bxb1 integrase-based site-specific integration for efficient, precise, and scalable protein tagging. Applied in human-induced pluripotent stem cells (hiPSCs), cancer cells and primary T cells, this system enables rapid generation of endogenously tagged proteins. By enhancing the nuclear localization signal of the catalytically superior eeBxb1 integrase and co-delivering a DNA-PK inhibitor, we achieved up to ∼90% integration efficiency at the ACTR10 locus in hiPSCs. ONE-STEP tagging is robust across loci and cell types and supports large DNA cargo integration, with efficiencies reaching 16.6% for a 14.4 kb construct. The method also enables multiplexed tagging of multiple proteins within the same cell and simultaneous CRISPR-based editing at secondary loci, such as gene knockouts or homology-directed repair. Importantly, we demonstrate successful application in primary T cells by targeting the T cell receptor locus while simultaneously knocking out B2M, a key step towards generating immune-evasive, off-the-shelf chimeric antigen receptor T cells. Additionally, we introduce a dual-cassette version of the method compatible with universal donor plasmids, allowing use of entirely off-the-shelf reagents. Together, these advances establish ONE-STEP tagging as a powerful tool for both basic and therapeutic genome engineering. © The Author(s) 2025. Published by Oxford University Press.
| Authors | Migliori V, Bruntraeger MB, Gyulev IS, Lichou F, Burgold T, Gitterman DP, Iwama S, Trinh AL, Washer SJ, Jones CP, Trynka G, Bassett AR |
|---|---|
| Journal | Nucleic acids research |
| Publication Date | 2025 Aug 11;53(15) |
| PubMed | 40823809 |
| PubMed Central | PMC12359036 |
| DOI | 10.1093/nar/gkaf809 |