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 Nucleus (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-core Cutting

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 clinical biopsy, rare cell populations, and other research scenarios with limited sample size.
• 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. Its design concept is to "move the experimental field from in vitro to the nucleus" to achieve in situ and specific cleavage of the target protein binding site. Its core innovation lies in using protein transposase Tn5 fusion protein (pA-Tn5) to directly fragment DNA and connect sequencing adapters at the antibody location.

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 complete the whole process from cells to sequencing library construction in a single day, and only 10,000 cells are needed to obtain high-quality data, achieving a triple breakthrough in sensitivity, efficiency, and background control.

Comparison Between CUT&RUN and Other Technologies

In addition to CUT&RUN, there are several other technologies that try to improve ChIP-seq, such as CUT&Tag (using protein A/G-Tn5 fusion protein to enhance antibody compatibility) and ChIC (using MNase).

• CUT&RUN and CUT&Tag are highly similar in principle, and the main difference lies in the activation mechanism and buffer liquid system of pA-Tn5, both of which are superior to the traditional ChIP-seq.
• Compared with ChIC, Tn5 transposase can integrate sequencing adapters while cutting, which simplifies the steps of library construction and improves the overall efficiency.

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 limit 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 magnetic beads coated with concanavalin A (ConA) for cell fixation can effectively exchange reagents and ensure the integrity of the nuclear structure throughout the whole experimental process. ConA specifically binds to glycosyl on the surface of the cell membrane, anchoring cells on magnetic beads to form a "bead-cell" complex, which is convenient for rapid and thorough buffer replacement through 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 can combine with cholesterol, form pores on the cell membrane, allow antibodies and pA-Tn5 to enter the nucleus, and keep the nuclear membrane relatively intact. 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: EDTA-free protease inhibitor mixture must be used, because the activity of pA-Tn5 depends on magnesium ion (Mg) in the subsequent step, and EDTA will chelate magnesium ion to completely inhibit enzyme activity.
    • 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-Tn5 Recruitment and Tagging: Engine for Precision Cutting

• A. Mechanism of pA-Tn5 transposase
  • a) PA-Tn5 is the executor of the CUT&RUN technology. It is a fusion protein, and its N-terminal is Protein A, which can bind the Fc segment of antibodies (mainly rabbit IgG and mouse IgG) with high affinity. Its C-terminal is Tn5 transposase with high activity. When pA-Tn5 was recruited to the target site of antibody binding through Protein A, in the presence of magnesium ion (Mg), Tn5 transposase was activated, and the surrounding DNA was double-stranded, and the pre-loaded sequencing linker was integrated at the end of the cut fragment. This feature of "cutting-joining" in one step greatly simplifies the library construction process.
• B. Exploration of optimum reaction conditions
  • a) PA-Tn5 concentration: Commercial pA-Tn5 is usually recommended to be used in the dilution range of 1:250 to 1:1000. Too high a concentration may increase nonspecific cleavage, while too low a concentration will lead to insufficient labeling efficiency of target sites. It is recommended to follow the manufacturer's instructions and optimize.
  • b) Magnesium ion concentration: Mg²⁺ is an essential cofactor of Tn5 activity, and the final reaction concentration is usually controlled at 5-10 mm.. Its concentration and reaction time jointly determine the degree of labeling.
  • c) Incubation temperature and time: The labeling reaction is carried out at 37°C, usually lasting for 1 hour. This step requires gentle mixing to prevent precipitation.
• C. Critical timing control
  • a) Tagging time is a variable that must be accurately controlled. The time is too short, resulting in insufficient effective fragments and low library yield. If the time is too long, it will lead to "over-digestion", produce short fragments, and increase background noise. For different target proteins (such as highly enriched histone modification vs sparsely distributed transcription factors), the optimal labeling time may need to be fine-tuned.
• D. Release and purification of DNA: recovery of target-specific fragments
  • a) Reaction termination strategy: After the labeling reaction is completed, the reaction must be terminated immediately and completely. It is mainly realized in two ways:
  • b) Adding EDTA: By chelating Mg, the enzyme activity of Tn5 was fundamentally terminated. The final concentration is usually 10 mM.
  • c) Adding SDS, a detergent, can denature protein, destroy the structure of pA-Tn5, and promote the chromatin complex to dissociate and release the cut DNA fragments. The working concentration is between 0.1% and 0.5%.
• E. Comparison of DNA extraction schemes
  • a) Protease K digestion: After adding the terminator, the system should be incubated at 55°C for at least 1 hour, and all proteins, including antibody and pA-Tn5, should be completely digested by protease K, so that DNA can be completely released.
  • b) Phenol-chloroform extraction+ethanol precipitation: This is a traditional and low-cost purification method, which can effectively remove protein and impurities, but the operation steps are complicated, and the recovery may be incomplete.
  • c) Purification of SPRI (Solid Phase Reversible Immobilization) Magnetic Beads: This is the more mainstream choice at present. SPRI magnetic beads can selectively bind DNA fragments in a specific length range in a concentration of PEG salt solution. After washing to remove impurities, DNA is eluted in a low salt buffer. The method is efficient, Qualcomm-intensive, and easy to automate, and can effectively remove the pollution of small fragments such as primer dimer.
  • d) Quality evaluation index: The purified DNA should be evaluated with a highly sensitive analytical instrument (such as Agilent Bioanalyzer or TapeStation). The DNA fragments produced by a successful CUT&RUN experiment should present a typical diffusion band of 100-700 bp, and the main peak is concentrated in the range of 200-500 bp. If a large number of fragments less than 100 bp appear, it may indicate excessive labeling or serious linker dimer pollution.

Similar performance using pA/MNase and pAG/MNase (Meers et al., 2019)

Library Amplification and Sequencing: Generating Library

• A. Amplification strategy
  • a) Because the amount of DNA recovered after tagging is extremely low, it is necessary to amplify it by PCR to obtain enough library for sequencing. However, PCR itself will introduce preference and repetitive sequences.
  • b) Limited cycle PCR: Usually 12-15 cycles of amplification are carried out. The goal of this cycle number is to maximize library diversity while minimizing amplification bias. Need to use high-fidelity DNA polymerase.
  • c) Double-ended index primer: PCR primer not only contains the sequence complementary to the Tn5 linker, but also contains the index sequence (i7 and i5) used for measuring instrument. Using a double-ended index allows Multiplex samples to be mixed in a single sequencing run and split by bioinformatics analysis, which greatly improves sequencing throughput and cost-effectiveness.
• 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 QCPR for accurate quantification, because QCPR only detects effective library molecules with complete linkers. Library concentration > 1 nM is usually required.
  • 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 linker dimer: In the electropherogram, the linker dimer peak at ~100 bp should be less than 5% of the total signal. Too high a dimer ratio will take up a lot of sequencing flux.
• C. Sequencing suggestion
  • a) Depth of sequencing: Depending on the research goal. For widely distributed histone modifications (such as H3K4me3, H3K27me3), 5 million to 20 million reads are usually enough. For transcription factors with narrow binding sites, more than 20 million reads may be needed to obtain sufficient signal coverage.
  • 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 clinical transformation and large-scale application, and a technical optimization breakthrough is urgently needed.

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.
• Multiplication and cellular-resolution prospect: Although standard CUT&RUN is a technology at the level of population cells, the combination of its principle and cellular-resolution sequencing technology has shown great potential, such as scCut&Tag, which is developing, and is expected to analyze the epigenetic state in cell 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 technology successfully solved many pain points of traditional ChIP-seq by combining targeted immunolocalization with the in vitro cleavage-ligation activity of Tn5 transposase, which provided a powerful tool for epigenetics research that was faster, more sensitive, with lower background, and more economical. 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.