What is CUT&RUN Sequencing and How Does It Compare to CUT&Tag?

Currently, epigenetic research is facing new challenges such as single-cell resolution, tracking dynamic processes, and compatibility with clinical samples. For example, the dynamic distribution of heterochromatin markers during embryonic development relies on the high-resolution characteristics of CUT&RUN, while the trace amounts in clinical biopsy samples are better suited to the low starting amount advantage of CUT&Tag. This article aims to systematically review the technical principles, core differences, and applicable scenarios of CUT&RUN and CUT&Tag, providing researchers with a reference for selecting appropriate methods based on target protein type, chromatin state, and experimental needs, and promoting epigenetic research towards higher precision and wider applicability.

Comparison of Principles and Technologies

CUT&RUN Sequencing: Principles and Features

CUT&RUN (Cleavage Under Targets and Release Using Nuclease) is an antibody-targeted chromatin analysis technique. It utilizes micrococcal nuclease (MNase) to cleave DNA near the target protein binding site and directly release the fragment for sequencing.

• Its core steps include:
  • Cell fixation and permeabilization: Live cells are bound using concanavalin A (ConA) magnetic beads, and permeabilization to the cell membrane is achieved using digitin, eliminating the need for formaldehyde cross-linking.
  • Antibody incubation: Specific antibodies target and bind to the target protein (such as histone modifiers or transcription factors) and are recruited to the chromatin binding site by the pA-MNase enzyme, a protein A/G fusion enzyme.
  • MNase cleavage: After Ca²⁺ activation, MNase cleaves the DNA flanking the target protein, releasing a fragment of approximately 300-500 bp that diffuses to the extranuclear region.
  • DNA Purification and Library Construction: DNA is directly purified, end-repaired, A-tailed, and ligated with sequencing adapters without the need for the sonication disruption step in traditional ChIP.
• Advantages:
  • High Resolution: Binding site localization is accurate to ±50 bp, making it particularly suitable for research on pioneer transcription factors (such as CTCF).
  • Heterochromatin Suitability: MNase enzymes have a small molecular weight and can efficiently cleave tightly packed heterochromatin regions (such as H3K9me3 modification sites), making it suitable for complex scenarios such as embryonic development.
  • Low Background Noise: Eliminates the need for sonication disruption, reducing interference from random DNA breaks.

Overall performance of "CUT&RUN on plate" (Miura M et al., 2020)

CUT&Tag: Technological Innovation and Simplified Process

CUT&Tag (Cleavage Under Targets and Tagmentation) is an improvement on CUT&RUN, using the fusion protein pA-Tn5 transposase to simultaneously complete DNA cleavage and sequencing adapter addition, significantly simplifying experimental procedures.

• Its workflow includes:
  • Antibody Incubation: Cells are immobilized using concanavalin A magnetic beads, and antibodies target and bind to the target protein.
  • Tn5 Transposition: After activating the transposase, DNA is directly cleaved at the target site and sequencing adapters are inserted.
  • Library Amplification: No end repair is required; sequencing libraries are generated directly through PCR amplification.
• Advantages:
  • Easy Operation: Eliminates cross-linking and sonication steps, shortening the experimental cycle to 1 day.
  • Low Starting Volume: Only 500-50,000 cells are needed, and even single-cell analysis is supported.
  • High Signal-to-Noise Ratio: Targeted cleavage reduces non-specific background, resulting in a data validity rate exceeding 80%.

Technical Differences Between CUT&RUN and CUT&Tag

Key Differences Analysis

• Nuclear Membrane Penetration and Chromatin State:
  • CUT&RUN fixes live cells using ConA magnetic beads, and MNase only cleaves target protein binding sites, providing strong penetration into heterochromatin (e.g., H3K9me3).
  • CUT&Tag relies on Tn5 transposase, which prefers open chromatin and may have insufficient coverage of heterochromatin, potentially leading to false negatives.
• Background Noise Control:
  • CUT&RUN uses precise MNase cleavage, releasing only target site DNA and resulting in very low background signals.
  • CUT&Tag's Tn5 transposase may cleave non-target regions, requiring optimization of antibodies and reaction conditions to reduce noise.

Key Differences: Application Scenarios and Limitations

(1) Detection of Heterochromatin Histone Modifications

• CUT&RUN is superior: MNase can cleave heterochromatin regions, while the Tn5 enzyme in CUT&Tag is difficult to effectively cleave closed chromatin due to steric hindrance. For example, research by Ruimin Xu et al. shows that the dynamic distribution of H3K9me3 in human embryonic development depends on CUT&RUN.

• Limitations of CUT&Tag: Tn5 enzyme prefers open chromatin, resulting in insufficient coverage of heterochromatin, which may lead to false negative results.

Schematic of sample preparation and CUT&RUN of histone modifications in human pre-implantation embryos (Xu R et al., 2022)

(2) Resolution of Transcription Factor Binding Sites

• CUT&RUN has higher resolution: MNase cleavage produces shorter DNA fragments with clearer binding site boundaries, making it particularly suitable for the study of pioneering transcription factors. For example, the enrichment efficiency of CUT&RUN-qPCR is 10 times higher than that of traditional ChIP-qPCR.

• Applicability of CUT&Tag: It works well for most transcription factors (such as RNA polymerase II), but the resolution is relatively low (approximately 200-500 bp).

(3) Experimental Complexity and Cost

• CUT&Tag is simpler: no library preparation and repair steps are required, making it suitable for high-throughput sample processing.

• CUT&RUN is slightly more expensive: additional optimization of MNase activity and adapter ligation is required, but the data quality is more stable.

Recommendations

Scenarios where CUT&RUN is preferred

High-resolution localization of transcription factor binding sites is required

For example, Miura M et al. used CUT&RUN technology to analyze the chromatin binding characteristics of RNA polymerase II (Pol II) in the human lung cancer cell line A549, revealing the unique biological significance of two differential binding structures of Pol II near the transcription start site (TSS) and short DNA footprints. The core findings are as follows: Using CUT&RUN (targeting micrococcal nuclease DNA cleavage recognition protein footprints) as a tool, the distribution of Pol II in A549 cells was analyzed:

• Long fragments (>270 bp): Corresponding to a bimodal distribution around the TSS (similar to classic ChIP results), representing paused Pol II (binding to complexes such as NELF, and being arrested after transcription initiation);

• Short fragments (< 120 bp): Accounting for approximately 5% of the total reads, but a clear peak can be identified at the TSS—this is a transient localization of Pol II before pause near the promoter (not detected by conventional ChIP). Some short fragments may also correspond to "abrupt initiation" (Pol II is released from the start site but cannot prolong transcription).

• Significance

• The unique footprints of short fragments (< 120 bp) reveal the transient localization of Pol II near the promoter, supplementing the blind spots of classic ChIP;

• The location of large footprints (long fragments) is correlated with transcriptional directionality, clarifying the functional value of different CUT&RUN footprints.

Working hypothesis on the Pol II footprints and Pol II pausing (Miura M et al., 2020)

Analysis of Heterochromatin Markers

For example, Yang H et al. used CUT&RUN to locate histone modifications across the entire genome and found that Brd4 deficiency led to a global increase in the enrichment levels of two active chromatin markers (H3K122ac: active transcription-related; H3K4me3: active promoter marker) in HSCs/HPCs. This change coincided with the upregulation of aging-related gene expression. This result reveals the mechanism by which Brd4 maintains HSC/HPC function and prevents aging by regulating histone modifications (inhibiting the over-enrichment of H3K122ac/H3K4me3).

Prioritized Scenarios for CUT&Tag

Limited sample size or requiring rapid experimental cycles

For example, Wu SJ et al. used scCUT&Tag technology to achieve single-cell chromatin landscape analysis of specific histone modifications (H3K27me3).

• Cell type differentiation and PcG landscape generation: scCUT&Tag analysis of H3K27me3 can differentiate human blood cell types, generating cell type-specific PcG landscapes from heterogeneous tissues.

• Tumor heterogeneity analysis:
  • Analysis of H3K27me3 in brain tumor patients before and after treatment (including relapsed samples) identified cell types and PcG activity heterogeneity in the tumor microenvironment;
  • Four cell types were identified in relapsed samples, revealing enrichment of the T1 cluster (similar to the T1 cluster in primary tumors). In drug-resistant cell clusters, the preneuronal genome (Verhaak_glioblastoma_proneural) was silenced, and the PcG landscape approached a stem-like state (non-terminally differentiated).

Studies on histone modifications in open chromatin regions

For example, Xie B et al. used CUT&Tag technology to detect the regulatory role of histone H3K18 lactylation (H3K18la) in bladder cancer, finding that:

• H3K18la is enriched in the promoter regions of numerous genes;

• CUT&Tag sequencing revealed that H3K18la-related genes are rich in multiple signaling pathways regulating tumorigenesis, indicating that H3K18la plays a crucial role in the progression of human bladder cancer.

• The LCN2 promoter region is enriched with H3K18la;

• Glycolysis inhibitors or circXRN2 overexpression can reduce the interaction between H3K18la and the LCN2 promoter, verifying the key regulatory role of H3K18la in LCN2 transcription (confirmed by subsequent ChIP experiments).

Summary

Both CUT&RUN and CUT&Tag have revolutionized chromatin research paradigms, but each has its own emphasis:

• CUT&RUN: Excels in high resolution and heterochromatin compatibility, making it the "gold standard" for complex epigenetic studies.

• CUT&Tag: Advantages include ease of use and low starting quantities, making it the preferred tool for routine histone modification analysis.

The two are complementary rather than interchangeable; researchers should choose flexibly based on the target protein type, sample characteristics, and experimental requirements.

People Also Ask

What is the difference between CUT&RUN and CUT&Tag?

CUT&RUN uses Ca2+-activated pAG-MNase to cleave the DNA while CUT&Tag uses Mg2+-activated pAG-Tn5 to cleave the DNA.

What is CUT&RUN sequencing?

CUT&RUN is an antibody-targeted chromatin profiling technique that cleaves DNA in situ within permeabilized nuclei, enabling high-resolution mapping of protein-DNA interactions with low background and minimal cell input.

What are the advantages of CUT&RUN over ChIP-seq?

CUT&RUN offers key advantages over ChIP-seq, including lower background noise, higher resolution, faster protocol, and significantly reduced cell input requirements.

What is the difference between CUT&Tag and ATAC seq?

ATAC-Seq enables researchers to detect the locations of open chromatin, nucleosomes, and occupied transcription factors using adapter-loaded Tn5. CUT&Tag reveals protein-chromatin interactions using a target antibody and adapter-loaded pA-Tn5.

References

1. Miura M, Chen H. CUT&RUN detects distinct DNA footprints of RNA polymerase II near the transcription start sites. Chromosome Res. 2020 Dec;28(3-4):381-393.
2. Xu R, Li S, Wu Q, Li C, Jiang M, Guo L, Chen M, Yang L, Dong X, Wang H, Wang C, Liu X, Ou X, Gao S. Stage-specific H3K9me3 occupancy ensures retrotransposon silencing in human pre-implantation embryos. Cell Stem Cell. 2022 Jul 7;29(7):1051-1066.e8.
3. Yang H, Sui P, Guo Y, Chen S, Maloof ME, Ge G, Nihozeko F, Delma CR, Zhu G, Zhang P, Ye Z, Medina EA, Ayad NG, Mesa R, Nimer SD, Chiang CM, Xu M, Chen Y, Yang FC. Loss of BRD4 induces cell senescence in HSC/HPCs by deregulating histone H3 clipping. EMBO Rep. 2023 Oct 9;24(10):e57032.
4. Wu SJ, Furlan SN, Mihalas AB, Kaya-Okur HS, Feroze AH, Emerson SN, Zheng Y, Carson K, Cimino PJ, Keene CD, Sarthy JF, Gottardo R, Ahmad K, Henikoff S, Patel AP. Single-cell CUT&Tag analysis of chromatin modifications in differentiation and tumor progression. Nat Biotechnol. 2021 Jul;39(7):819-824.
5. Xie B, Lin J, Chen X, Zhou X, Zhang Y, Fan M, Xiang J, He N, Hu Z, Wang F. CircXRN2 suppresses tumor progression driven by histone lactylation through activating the Hippo pathway in human bladder cancer. Mol Cancer. 2023 Sep 8;22(1):151.