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AbstractWe present a novel, versatile genome editing method termed ONE-STEP tagging, which combines CRISPR¨CCas9-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. Graphical Abstract Graphical Abstract

Human immunodeficiency virus (HIV) remains a global health challenge with major research efforts being directed toward the unmet needs for a vaccine and a safe and scalable cure. Antiretroviral therapy (ART) suppresses viral replication but does not cure infection and so requires lifelong adherence. HIV-specific CD8+ T-cell responses play a crucial role in long-term HIV control as demonstrated in elite controllers, highlighting their potential in HIV cure strategies. Various HIV mouse models-including the human-hematopoietic stem cell (Hu-HSC) mouse, the bone marrow, liver, and thymus (BLT) mouse, and the human peripheral blood leukocyte (Hu-PBL) mouse-have deepened the understanding of HIV dynamics and facilitated the development of therapeutics. We developed the HIV participant-derived xenograft (HIV PDX) mouse model to enable long-term in vivo evaluation of bona fide autologous T-cell mechanisms of HIV control, including the antiviral activity of primary memory CD8+ (mCD8+) T cells taken directly from people with or without HIV, as well as testing potential immunotherapies. Additionally, this model faithfully recapitulates virus escape mutations in response to sustained CD8+ T-cell pressure, enabling the assessment of strategies to curb virus escape. In this model, NSG mice are engrafted with purified memory CD4+ (mCD4+) cells and infected with HIV; then, they receive autologous CD8+ T cells or T-cell products. Key advantages of this model include the minimization of graft-versus-host disease (GvHD), which severely limits peripheral blood mononuclear cell (PBMC) or total CD4-engrafted mice, the ability to evaluate long-term natural donor-specific T-cell responses in vivo, and the lack of use of human fetal tissues required for most humanized mouse models of HIV. Key features ? Long-term evaluation of bona fide autologous T cells. ? Evaluation of immunomodulating drugs and T-cell products. ? The protocol requires access to a BSL2+ tissue culture room, BSL2+ animal facility, and 6+ weeks to complete.

PurposeCD4+ effector T cells (Teffs) play a key role in immune responses by infiltrating the sites of inflammation and modulating local leukocyte activity. In turn resident immune cells shape their response. This study aimed to investigate the influence of mast cells (MCs) on Teff biological responses.MethodsThis study examined human MC-Teff interactions, focusing on how MCs shape Teff responses. Flow cytometry, qRT-PCR, and cytokine assays were used to analyze the impact of primary human MCs on the Teff phenotype and function. MC-Teff crosstalk within Crohn¡¯s disease patient tissues was assessed using confocal microscopy and advanced image analysis.ResultsMCs promoted the differentiation of Th17 cells, particularly the inflammatory Th17.1 subset, that secretes IFN-¦Ã and GM-CSF. This differentiation was driven by the PGE2 and IL-1¦Â axis. Additionally, MCs disrupted the phenotype and impaired the suppressive function of regulatory T cells (Tregs) through PGE2, skewing the Th17/Treg balance. The analysis of biopsies from patients with Crohn¡¯s disease indicated that this MC/Teff crosstalk may play a role in the pathogenesis of auto-inflammatory processes.ConclusionMCs influence CD4+ T cell responses by fostering pro-inflammatory Th17 differentiation while impairing Treg function. This interaction underpins a Th17/Treg imbalance, which is significant in auto-inflammatory diseases such as Crohn¡¯s disease, positioning MCs as critical drivers of disease pathogenesis. Plain language summaryThe immune system functions through complex interactions between different types of cells to protect the body from harm. CD4+ T cells are crucial in managing inflammation by directing other immune cells. Mast cells, found in tissues such as the skin and gut, are among the first to respond to potential threats and can influence T cell behavior. This study examined how mast cells and T cells interact in vitro, particularly in Crohn¡¯s disease, a condition in which the immune system causes gut inflammation.We used laboratory techniques to study how mast cells affect T cells, examining their behavior and communication. We also studied tissue samples from individuals with Crohn¡¯s to determine how these interactions occur in real life.We found that mast cells encourage the development of inflammatory T cells called Th17, particularly a type known as Th17.1, which produce strong inflammatory signals. This process relies on specific molecules such as PGE2 and IL-1¦Â. Simultaneously, mast cells weaken the function of regulatory T cells (Tregs), which normally help control inflammation. This leads to an imbalance between Th17 cells and Tregs, tipping the immune system toward excessive inflammation. In tissue samples from Crohn¡¯s disease patients, we found evidence of this imbalance, suggesting that mast cells play a major role in driving harmful inflammation.These findings help us understand why inflammation becomes uncontrollable in diseases such as Crohn¡¯s disease. They also suggest that targeting mast cells could be a promising strategy for new treatments.