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Af-CUT&Tag: A Sensitive and Antibody-Free Chromatin Profiling Method Using Genetically Encoded Tags and High-Affinity Binders Fused to Tn5

ABSTRACT: Conventional chromatin profiling techniques are often limited by antibody availability and performance. Here, we introduce Af-CUT&Tag, an antibody-free method that overcomes these limitations by using CRISPR-integrated peptide tags (HiBiT/ALFA-tag) targeted by engineered binders (LgBiT/NbALFA) fused to a Tn5 transposase. Af-CUT&Tag eliminates antibody dependence, achieving superior specificity and sensitivity with as few as 500 cells. This method outperforms antibody-based counterparts in signal-to-noise ratio, promoter enrichment, and library quality, while also enabling single-cell resolution (scAf-CUT&Tag). Applying Af-CUT&Tag to Hippo effectors (YAP1/TAZ) during liver regeneration revealed dynamic chromatin remodeling, including YAP1/TAZ-mediated control of lipid metabolism (e.g., Lpin1, Fasn) and heme clearance (Hpx, Trf). We further identify miR-122 as a critical regulator of these processes, impacting liver regeneration. The versatility of Af-CUT&Tag in cell lines, bulk tissues, and single nuclei establishes it as a transformative tool for studying gene regulation in development, disease, and regeneration. Keywords: Antibody-Free CUT&Tag; Chromatin Binding; Epigenetic Profiling; Peptide-binder; Single-cell analysis

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE283704 | GEO | 2024/12/15

REPOSITORIES: GEO

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Af-CUT&Tag: A Sensitive and Antibody-Free Chromatin Profiling Method Using Genetically Encoded Tags and High-Affinity Binders Fused to Tn5

Project description:Conventional chromatin profiling techniques are often limited by antibody availability and performance. Here, we introduce Af-CUT&Tag, an antibody-free method that overcomes these limitations by using CRISPR-integrated peptide tags (HiBiT/ALFA-tag) targeted by engineered binders (LgBiT/NbALFA) fused to a Tn5 transposase. Af-CUT&Tag eliminates antibody dependence, achieving superior specificity and sensitivity with as few as 500 cells. This method outperforms antibody-based counterparts in signal-to-noise ratio, promoter enrichment, and library quality, while also enabling single-cell resolution (scAf-CUT&Tag). Applying Af-CUT&Tag to Hippo effectors (YAP1/TAZ) during liver regeneration revealed dynamic chromatin remodeling, including YAP1/TAZ-mediated control of lipid metabolism (e.g., Lpin1, Fasn) and heme clearance (Hpx, Trf). We further identify miR-122 as a critical regulator of these processes, impacting liver regeneration. The versatility of Af-CUT&Tag in cell lines, bulk tissues, and single nuclei establishes it as a transformative tool for studying gene regulation in development, disease, and regeneration. Keywords: Antibody-Free CUT&Tag; Chromatin Binding; Epigenetic Profiling; Peptide-binder; Single-cell analysis

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