ArticleNucleic acids research2025
ONE-STEP tagging: a versatile method for rapid site-specific integration by simultaneous reagent delivery.
Article in Nucleic acids research, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 5 papers.
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Who cites it
5 citing papers in PubMed.
- Gene editing using large insertions.Nature biotechnology · 2026Article
- Large serine recombinase-mediated gene insertion for high-throughput screens: advantages, design principles, and applications.Nucleic acids research · 2026Review
- Retargeted serine integrases for one-step, precise integration of large DNA sequences in human cells.Nature biotechnology · 2026Article
- Gene-sized editing for the therapy of genetic diseases.Functional & integrative genomics · 2026Review
- Next-generation T cell immunotherapies engineered with CRISPR base and prime editing: challenges and opportunities.Nature reviews. Clinical oncology · 2025Review
Corrections and comments
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Authors and funding
12 authors.
Funding
Abstract
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.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.