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<p>Conventional chromatin profiling techniques are often limited by antibody availability and performance. Here, we introduce Af-CUT&amp;Tag, a target antibody-free method that overcomes these limitations by using CRISPR-integrated peptide tags (HiBiT/ALFA-tag) recognized by engineered binders (LgBiT/NbALFA) fused to a Tn5 transposase. Af-CUT&amp;Tag eliminates dependence on traditional target antibodies, achieving robust specificity and sensitivity with as few as 500 cells. It provides high-quality chromatin profiles, with improved signal-to-noise ratios and library quality compared with conventional antibody-based counterparts, while also enabling single-cell resolution (scAf-CUT&amp;Tag). Applying Af-CUT&amp;Tag to Hippo effectors (YAP1/TAZ) during liver regeneration reveals dynamic chromatin remodeling, including YAP1/TAZ-mediated control of lipid metabolism (e.g., <i>Lpin1, Fasn</i>) and heme clearance (<i>Hpx, Trf</i>). We further identify miR-122 as a critical regulator of these processes, impacting liver regeneration. The versatility of Af-CUT&amp;Tag in cell lines, bulk tissues, and single nuclei establishes it as a powerful tool for studying gene regulation in development, disease, and regeneration.</p>

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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

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年1月17日

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  • Xindong Wang,
  • Xusheng Deng,
  • Lu Qiu,
  • Junjia Liu,
  • Hengxiang Shen,
  • Haoran Du,
  • Weijie Li,
  • Linxuan Song,
  • Wenhui Deng,
  • Xiaoning Dong,
  • Yi Han,
  • Benchao Liu,
  • Jialiang Huang,
  • Zengpeng Li,
  • Yongyou Zhang

摘要

Conventional chromatin profiling techniques are often limited by antibody availability and performance. Here, we introduce Af-CUT&Tag, a target antibody-free method that overcomes these limitations by using CRISPR-integrated peptide tags (HiBiT/ALFA-tag) recognized by engineered binders (LgBiT/NbALFA) fused to a Tn5 transposase. Af-CUT&Tag eliminates dependence on traditional target antibodies, achieving robust specificity and sensitivity with as few as 500 cells. It provides high-quality chromatin profiles, with improved signal-to-noise ratios and library quality compared with conventional antibody-based counterparts, while also enabling single-cell resolution (scAf-CUT&Tag). Applying Af-CUT&Tag to Hippo effectors (YAP1/TAZ) during liver regeneration reveals 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 powerful tool for studying gene regulation in development, disease, and regeneration.