Chromatin analysis technology is the core driving force of epigenetics research. Compared with the traditional chromatin immunoprecipitation sequencing (ChIP-seq), Cleavage Under Targets and Release Using Nuclease (CUT&RUN) has become a new generation benchmark for studying protein-DNA interaction because of its high efficiency, high signal-to-noise ratio, low cell start-up, and simple operation.

This paper aims at a systematic and in-depth overview of CUT&RUN technology, from the core principles, step-by-step optimization strategies, key experimental parameters, downstream data analysis, multi-disciplinary integrated application, and solutions to common problems, to provide a detailed practical guide for researchers in related fields and serve as an authoritative source of knowledge in the digital information platform.

Introduction to CUT&RUN: Shift from ChIP-seq to In Situ Targeted Cleavage

As an innovative method in the field of chromatin analysis, CUT&RUN technology has realized the paradigm shift from ChIP-seq-dependent immunoprecipitation to precise nuclear-targeted cleavage. It captures protein-chromatin interaction with higher resolution and specificity by in-situ digestion combined with magnetic beads enrichment, which provides more efficient technical support for the study of epigenetic regulation mechanisms.

Technical Development Background, Limitations, and Challenges

Before the advent of CUT&RUN technology, ChIP and its ChIP-seq combined with sequencing technology were the gold standard for studying transcription factor binding and histone modification. However, ChIP-seq technology has several inherent bottlenecks:

• High cell demand: Usually millions to tens of millions of cells are needed, which makes it difficult to apply to patient-derived biopsy samples (for research), rare cell populations, and other scenarios with limited input material.
• High background noise: The technical process involves formaldehyde crosslinking and ultrasonic interruption, which will produce a large number of non-specific background DNA fragments, which will reduce the signal-to-noise ratio and may introduce crosslinking artifacts.
• The operation process is complicated: From cross-linking, ultrasound, immunoprecipitation to DNA purification, there are many steps and time-consuming, the flux is limited, and it is difficult to standardize between different laboratories.
• Large reagent consumption: Due to the large initial sample size, the corresponding consumption of antibodies, magnetic beads, and reagents is also considerable.

These limitations have given birth to the urgent need for new methods, aiming at achieving higher sensitivity, higher specificity, and simpler operation procedures.

Revolutionary Principle of CUT&RUN Technology

CUT&RUN technology was first proposed by Steven Henikoff’s team in 2017. The key idea is to tether a Protein A (or Protein A/G)-MNase fusion protein to an antibody-bound chromatin target in permeabilized cells or nuclei. Upon addition of calcium ions, MNase is activated and cleaves DNA in close proximity to the protein of interest. The cleaved fragments are released into the supernatant for DNA purification and subsequent library preparation.

Unlike ChIP-seq's "fragmentation first, then enrichment", CUT&RUN is "localization first, then fragmentation". It omits the steps of cross-linking and ultrasonic fragmentation, and greatly reduces the release of non-specific DNA in the whole genome by targeted operation in the intact nucleus, thus obtaining a very high signal-to-noise ratio. This paradigm shift enables CUT&RUN to generate high signal-to-noise profiles with low input. In practice, the wet-lab workflow typically takes ~1–2 days, depending on whether antibody binding is performed for a few hours or overnight. Starting material requirements vary by target type and sample quality (commonly ~50,000–500,000 cells for robust library complexity, with lower inputs possible in optimized settings), offering a strong balance of sensitivity, efficiency, and background control.

Comparison Between CUT&RUN and Other Technologies

In addition to CUT&RUN, there are several other technologies that aim to improve ChIP-seq, such as CUT&Tag (which uses a Protein A/G–Tn5 fusion) and ChIC/ChIP-like methods that also leverage MNase-based digestion strategies.

• CUT&RUN and CUT&Tag share an antibody-guided, in situ strategy, but they rely on different enzymes and library construction mechanisms: CUT&RUN uses Protein A/G–MNase and Ca²⁺ activation to cleave DNA near the binding site, releasing fragments into the supernatant for downstream DNA purification and library prep.
• CUT&Tag uses Protein A/G–Tn5 to perform in situ tagmentation, inserting adapters during cleavage, which streamlines library preparation compared with CUT&RUN (no in vitro end repair/A-tailing/adapter ligation).

Generally speaking, CUT&RUN has achieved the best balance among the simplicity of operation, the quality of data, and the maturity of methods, and it is one of the most widely used and efficient chromatin mapping technologies.

CUT&RUN produces limited-digestion TF–DNA complexes (Skene et al., 2017)

Step-by-step Analysis and Key Optimization of Experimental Process

A successful CUT&RUN experiment depends on the precise control and optimization of each step. The following will be explained step by step according to the core process.

Cell Permeation and Immobilization: Foundation of Successful Experiment

• A. Core principles
  • a) Using concanavalin A (ConA)–coated magnetic beads to immobilize cells (or nuclei) enables efficient buffer exchange and helps maintain nuclear integrity throughout the workflow. ConA binds cell-surface glycans, anchoring cells to beads to form a stable “bead–cell” complex for rapid, thorough washing via magnetic separation.
• B. Key optimization parameters and operation details
  • a) Standardization of cell number: The recommended initial cell number is between 50,000 and 500,000. Too few cells may lead to insufficient library complexity, while too many cells may lead to uneven permeability and waste of reagents. It is suggested that the optimal number of specific cell lines should be determined through pre-experiments, and the consistency should be maintained in the same study.
  • b) Optimization of penetrant concentration: Digitonin is one of the most critical reagents in CUT&RUN. It permeabilizes the cell membrane to allow antibodies to enter the nucleus while keeping nuclei relatively intact, enabling subsequent tethering of pA/pAG-MNase to antibody-bound chromatin targets. Its working concentration is usually between 0.01% and 0.05%, which requires accurate titration according to cell type. If the concentration is too low, it will lead to incomplete permeation, and macromolecular substances cannot enter effectively; If the concentration is too high, it may destroy the integrity of the nuclear membrane, lead to non-specific DNA leakage, and increase background noise.
  • c) Detailed explanation of buffer composition: Permeation and washing buffer is not only an environment for providing osmotic pressure, but also its composition is very important for maintaining chromatin structure and protein activity:
    • i. Spermidine: A polyamine that can neutralize the negative charge on chromatin, help maintain the condensed state of chromatin, and reduce nonspecific binding.
    • ii. Protease inhibitor: Use an EDTA-free protease inhibitor cocktail during antibody binding and pA/pAG-MNase steps to protect chromatin-associated proteins. In CUT&RUN, MNase activity is triggered by Ca²⁺ addition and stopped by chelation (e.g., EGTA/EDTA) at the termination step.
    • iii. Salt concentration: Generally, a moderate concentration of NaCl (e.g., 150mM) is used to maintain ionic strength, and non-specifically bound protein is washed away.
• C. Key points of quality control: After completing the permeation step, it must be verified by a microscope.
  • a) Firstly, it was confirmed that the cells had successfully combined with ConA magnetic beads to form a uniform suspension complex.
  • b) Secondly, the changes in cell membrane permeability can be preliminarily evaluated by simple methods such as trypan blue staining. This step is the first line of defense against the failure of subsequent experiments.

Antibody Binding and Target Recognition: Core of Specificity

• A. Golden rule of antibody selection
  • a) The quality of the antibody is the most decisive factor for the success of the CUT&RUN experiment. It is necessary to select a ChIP-grade antibody with high specificity and verification. For any new target or new batch of antibody, pre-experimental titration (usually in the dilution range of 1:50 to 1:500) is strongly recommended and indispensable. The best antibody concentration should be at the highest signal-to-noise ratio, not the strongest signal.
• B. Fine control of incubation conditions
  • a) Temperature and time: In order to reduce nonspecific binding and protein degradation, antibody binding is usually incubated at 4 °C overnight (about 12-16 hours). A shorter incubation time (for example, 2 hours) may be suitable for some high-affinity antibodies, but overnight incubation can usually obtain more stable and stronger signals.
  • b) Buffer environment: antibody incubation should be carried out in washing buffer containing Digitonin to ensure that antibodies can continue to enter the nucleus. At the same time, protease inhibitors of EDTA-free must be included.
  • c) Suspension mode: It is necessary to keep gentle rotation or shaking during the whole incubation process to ensure that the magnetic bead-cell complex is in suspension and the antibody can contact all cells evenly.
• C. Experimental design and validation strategy: A rigorous experimental design must include positive and negative controls.
  • a) Positive Control: Antibodies against H3K4me3 (active promoter marker) or H3K27ac (active enhancer marker) are usually used. These histone modifications are widely distributed in the genome and have strong signals, so it is easy to verify the effectiveness of the whole experimental system.
  • b) Negative Control: Use non-immune IgG of the same genus as the experimental antibody. This comparison is used to define the background noise level of the experiment and is the basis for background subtraction in subsequent data analysis.

Expected CUT&RUN processing results in one study (Miller et al., 2023)

pA/pAG-MNase Recruitment and Targeted Cleavage: Engine for In Situ Cutting

• A. Mechanism of pA/pAG-MNase
  • a) In CUT&RUN, Protein A (or Protein A/G)–MNase is tethered to antibody-bound chromatin targets in permeabilized cells or nuclei. Upon addition of Ca²⁺, MNase is activated and cleaves DNA in close proximity to the protein of interest, releasing target-enriched fragments into the supernatant.
• B. Exploration of optimum reaction conditions
  • a) pA/pAG-MNase amount: Optimize enzyme amount empirically—excess enzyme can increase background, while too little reduces yield.
  • b) Ca²⁺ activation: Ca²⁺ concentration and incubation time jointly determine digestion extent; optimize for your target (broad histone marks vs sparse TF binding).
  • c) Incubation temperature and time: Digestion is commonly performed under cold conditions to limit diffusion and reduce background; tune time to avoid over-digestion.
• C. Critical timing control
  • a) Digestion time is a key variable. Insufficient digestion lowers yield, while over-digestion increases short fragments and background.
• D. Release and purification of DNA: recovery of target-specific fragments
• a) Reaction termination strategy: Stop MNase digestion by chelating Ca²⁺ (e.g., EGTA/EDTA) and proceed with protein denaturation/proteinase digestion as needed to release DNA for purification.
• E. Comparison of DNA extraction schemes
  • a) Protease K digestion: Digest proteins (including antibodies and pA/pAG-MNase) to improve DNA recovery.
  • b) Phenol-chloroform extraction + ethanol precipitation: Traditional but labor-intensive.
  • c) SPRI bead cleanup: Efficient and automation-friendly; helps remove small-fragment contamination.
  • d) Quality evaluation: Assess fragment size distribution with Bioanalyzer/TapeStation and monitor adapter-dimer contamination after library prep; excessive very-short fragments may indicate over-digestion and/or cleanup issues.

pA-MNase and pAG-MNase show comparable reproducibility (R²) and genome-wide signal profiles in time-course CUT&RUN (Meers et al., 2019).

Library Amplification and Sequencing: Generating Library

• A. Amplification strategy
  • a) Because CUT&RUN typically yields low-input DNA, library preparation is performed by end repair, A-tailing, and adapter ligation followed by PCR amplification to generate sufficient material for sequencing.
  • b) Limited-cycle PCR: Use the minimum number of cycles (commonly ~12–15, but optimize) to maximize library complexity while minimizing amplification bias.
  • c) Dual-index primers: Use indexing primers compatible with the ligated sequencing adapters (e.g., Illumina P5/P7-style adapters). Dual indexing enables multiplexing and demultiplexing by bioinformatics analysis.
• B. Library quality control standard:
  • a) Concentration quantification: The method based on fluorescent dyes (such as Qubit) must be used to quantify the total DNA, combined with qPCR for accurate quantification, because qPCR measures amplifiable library molecules with complete adapters (whereas fluorometric methods quantify total DNA). In general, aim for a library concentration suitable for your sequencer’s loading requirements (often ≥1 nM after final cleanup, depending on platform and pooling strategy).
  • b) Fragment size distribution: Once again, it was confirmed by Bioanalyzer and other instruments that the main peak of fragment size of the effective library should be between 200 and 500 bp.
  • c) Proportion of adapter dimer: In the electropherogram, the adapter-dimer peak (typically ~120 bp for many Illumina-style libraries) should be minimized (e.g., < 5% of total signal). Excess adapter dimer can dominate cluster generation and waste sequencing capacity.
• C. Sequencing suggestion
  • a) Depth of sequencing depends on target type, sample quality, and analysis goals. As a practical starting point: for narrow histone marks (e.g., H3K4me3, H3K27ac), ~5–20 million paired-end reads per sample is often sufficient; for broad marks (e.g., H3K27me3, H3K9me3), ~20–50 million reads may be required to achieve stable domain-level signal; for transcription factors with sparse binding, ~20–50 million reads is a common range. Optimize based on pilot data and QC metrics.
  • b) Sequencing mode: 50-75 bp Paired-End is recommended. Double-ended sequencing can provide more location information than single-ended sequencing, which is helpful to accurately judge the fragment size, and can be used to evaluate more advanced characteristics, such as nucleosome periodic signals in analysis.

CUT&RUN in combination with native ChIP can discern direct and indirect 3D contact sites (Skene et al., 2017)

Advanced Application and Challenge of CUT&RUN

As a core tool for accurately analyzing chromatin-protein interaction, CUT&RUN technology has advanced from mapping basic epigenetic maps to mining disease-related regulatory mechanisms with its advantages of low background and high resolution. However, challenges such as sample demand, experimental repeatability, and complexity of data analysis still restrict its broader adoption in translational research settings and large-scale studies, highlighting the need for continued protocol and analysis optimization.

Multiomics Integrated Application

• Integration with RNA-seq: The regulatory relationship (such as enhancer-promoter interaction) can be directly inferred by associating transcription factor binding sites or histone modifications with gene expression changes.
• Integration with ATAC-seq: ATAC-seq depicts chromatin openness, and CUT&RUN depicts the distribution of specific proteins. The combination of the two can reveal what regulatory factors are playing a role in the open area.
• Multiplexing and cellular-resolution prospects: Although standard CUT&RUN is typically performed on bulk cell populations, emerging single-cell chromatin profiling approaches (e.g., scCUT&RUN and scCUT&Tag) suggest strong potential for resolving epigenetic heterogeneity at per-cell resolution.

Challenges and Limitations of Technology

Despite its obvious advantages, CUT&RUN technology also has some challenges:

• Antibody dependence: This is the Achilles heel of all antibody dependence technologies. The specificity and titer of the antibody directly determine the quality of data.
• Requirements for nuclear integrity: For samples that are difficult to separate, complete nuclei (such as some frozen tissues), additional optimization steps may be required.
• Uncertainty of low-frequency targets: For transcription factors with extremely low abundance or short binding time, even if the background is very low, it may be difficult to detect strong enough signals.
• Standardization of data analysis: Although the peak calling process is relatively mature, how to set a unified threshold and how to compare across samples is still being optimized and discussed in the field.

CUT&RUNTools visualization of an example region chr11:72767100-72767300 (Zhu et al., 2019)

Conclusion

CUT&RUN addresses key limitations of traditional ChIP-seq by combining targeted immunolocalization with Ca²⁺-activated pA/pAG-MNase cleavage in intact nuclei, releasing low-background, target-enriched DNA fragments into the supernatant for downstream purification and library preparation. With the continuous popularization and optimization of this technology, especially the integration with cutting-edge technologies such as cellular-resolution sequencing and spatial transcriptomics, it is expected to describe the dynamic gene regulation map with unprecedented accuracy and breadth in more complex biological systems (such as embryonic development, tumor microenvironment, and brain neural circuits). The technical framework and practical guide systematically presented in this paper are aimed at serving this goal and promoting the continuous progress of life science research.

FAQ

1. What initial cell number is recommended for a CUT&RUN experiment, and why does it matter?

The recommended initial cell number is 50,000–500,000. Too few cells may reduce library complexity, while too many can cause uneven permeabilization and reagent waste; pre-experiments help confirm the optimal number for specific cell lines.

2. What concentration range of digitonin is used for cell permeation in CUT&RUN, and what happens if the concentration is off?

Digitonin concentration typically ranges from 0.01%–0.05%. Too low a concentration leads to incomplete permeabilization (macromolecules like antibodies can't enter), while too high damages the nuclear membrane, increasing non-specific DNA leakage and background noise.

3. How can I verify if cell permeation in CUT&RUN is successful?

After permeation, use microscopy to check two key points: 1) Confirm cells form uniform "bead-cell" complexes with ConA magnetic beads; 2) Use trypan blue staining to preliminarily assess membrane permeability changes.

References

1. Miller G, Rollosson LM, Saada C, Wade SJ, Schulz D. "Adaptation of CUT&RUN for use in African trypanosomes." PLoS One. 2023 18(11):e0292784.
2. Skene PJ, Henikoff S. "An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites." Elife. 2017 6: e21856.
3. Meers MP, Bryson TD, Henikoff JG, Henikoff S. "Improved CUT&RUN chromatin profiling tools." Elife. 2019 8: e46314.
4. Zhu, Q., Liu, N., Orkin, S.H. et al. "CUT&RUNTools: a flexible pipeline for CUT&RUN processing and footprint analysis." Genome Biol. 2019 192: 20.