Cut-and-Tag

CUT&Tag / CUT&RUN (Cleavage Under Targets and Tagmentation / Release Using Nuclease)

一行要約

CUT&Tag / CUT&RUN は ChIP-seq の crosslinking + sonication を回避し、in situ で抗体に protein A/G-Tn5 (CUT&Tag) または protein A/G-MNase (CUT&RUN) を tether させて標的近傍のみを cleavage / tagmentation する手法。low input (細胞 100-100,000) で TF / histone modification を高 signal-to-noise で profile でき、ChIP-seq の細胞数要件を 10-100 分の 1 に削減。臨床検体の retrospective 解析や single-cell 拡張 (scCUT&Tag) で SCLC subtype TF / T cell exhaustion epigenetic 解析を可能にした。

原理

(1) 細胞 / 核を Concanavalin-A magnetic bead 上に固定。(2) primary 抗体 (TF or histone modification 特異的) で標的 binding。(3) CUT&Tag: secondary 抗体不要、protein A/G-Tn5 fusion を結合 → in situ tagmentation で sequencing adapter 挿入 → PCR amplification → 直接 library 化。CUT&RUN: protein A/G-MNase fusion → calcium 添加で MNase 活性化 → 標的近傍 DNA cleavage → 上清回収 → end repair / library prep。(4) Illumina sequencing → MACS2 / SEACR で peak calling。

scCUT&Tag は同じ原理を droplet single-cell に拡張、per-cell histone modification (H3K27ac / H3K4me3 / H3K27me3) を取得。MulTI-Tag は同一細胞で複数 modification を同時 profiling。

主要エビデンス / 適用領域

• SCLC TF profiling: ASCL1 / NEUROD1 / POU2F3 binding を low-input で patient-derived 検体に適用
• T 細胞 exhaustion epigenetic: TOX / NR4A1 / EOMES の binding を tumor-infiltrating T 細胞で profile
• Drug-tolerant persister chromatin: EGFR-TKI 持続細胞の H3K27me3 / H3K4me1 redistribution
• Clinical biopsy chromatin: small biopsy / FNA から TF binding map、ChIP-seq で要 1-10M 細胞 → CUT&Tag で 10K 以下対応
• Histone modification atlas: pan-cancer Encode-style atlas を low-input で構築

適用分野と限界

• 強み: low-input, high signal-to-noise, simple protocol (1-day ChIP), single-cell 拡張可能, FFPE 対応 (CUT&Tag-FFPE 開発中)
• 限界: TF binding は antibody 品質依存, scCUT&Tag は data sparsity, peak calling pipeline が ChIP-seq よりも standardization 進行中, antibody → Tn5 tether の steric bias 報告あり

Open Questions

• Single-cell multi-modification (MulTI-Tag) の routine 化
• FFPE CUT&Tag の clinical retrospective 適用
• Spatial CUT&Tag (slide-CUT&Tag) の解像度向上
• Quantitative comparison: ChIP-seq との absolute 一致性検証

関連エンティティ

• 補完手法: ChIP-seq / scATAC-seq / ATAC-seq / scRNA-seq / Multiome
• 関連遺伝子: ASCL1 / NEUROD1 / POU2F3 / MYC / EZH2
• 関連概念: Lineage-plasticity / T-cell-exhaustion / Epigenetic-therapy-resistance / NE-non-NE-transition

関連論文

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• Fu et al. CancerCell 2024 — Overcoming tyrosine kinase inhibitor resistance in lung cancer brain metastasis with CTLA4 blockade

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Basic-Others

• Zhang et al. GenomeBiol 2008 — Model-based analysis of ChIP-Seq (MACS)
• Laisne et al. NatRevCancer 2025 — Epigenomic heterogeneity as a source of tumour evolution
• Goto et al. Nature 2024 — SOX17 enables immune evasion of early colorectal adenomas and cancers

Clinical-NSCLC-Driver-Other

• Tong et al. CancerCell 2024 — Adeno-to-squamous transition drives resistance to KRAS inhibition in LKB1 mutant lung cancer