CUT&Tag: 4 Dimensions to Revolutionary Epigenomic Profiling

In the field of epigenetics, the dynamic interaction between proteins and DNA is the core link to analyzing gene expression regulation, cell differentiation, and disease mechanisms. From the early chromatin immunoprecipitation (ChIP) technology to the ChIP-seq technology combined with high-throughput sequencing, and then to the CUT&Tag technology that emerged in recent years, each technical iteration aims to break through the limitations of the preamble method and achieve higher resolution, lower sample requirements, and more accurate protein-DNA interaction detection.

Although the traditional ChIP-seq has occupied the position of gold standard for a long time, it faces many bottlenecks in practical application. CUT&Tag technology circumvents the core defects of ChIP-seq through innovative in-situ targeted cutting strategy, and provides a new path for the research of rare samples and high sensitivity requirements.

This article examines four aspects: (1) the progression from ChIP-seq to CUT&Tag; (2) the core mechanism of CUT&Tag; (3) its advantages; and (4) representative application scenarios, to support assay selection in epigenetics research. We provide details below.

From ChIP-seq to CUT&Tag: The Evolutionary Leap

Epigenetic studies of protein-DNA interactions have advanced rapidly. ChIP-seq, long a gold standard, faces drawbacks like cross-linking artifacts and high cell input. CUT&Tag emerges as a transformative alternative, enabling efficient, low-input, and high-resolution profiling, marking a pivotal evolutionary leap in the field.

Core Principle and Technical Bottleneck of ChIP-seq

ChIP-seq is a classic technology based on immunoprecipitation enrichment-high-throughput sequencing. Its core principle is to capture the DNA fragments bound to the target protein through specific antibodies, and then locate the positions of these fragments on the genome through sequencing, so as to draw the protein-DNA interaction map in the whole genome.

Although ChIP-seq has been widely used in the research of histone modification and transcription factor binding, its application in more scenarios is limited by inherent defects brought by technical principles. The main bottlenecks include:

• Signal deviation caused by crosslinking and fragmentation: Formaldehyde crosslinking may trigger nonspecific protein-DNA binding and increase background noise. Ultrasonic fragmentation is difficult to accurately control the fragment length, and may destroy the epitope of the target protein, resulting in the ineffective binding of antibodies, especially for structurally sensitive transcription factors.
• Demand for high sample size: ChIP-seq usually needs 100,000 to millions of cells as starting materials due to the loss of samples during immunoprecipitation, which cannot meet the research needs of rare cells (such as circulating tumor cells, CTCs, and neural stem cells) or clinical micro samples (such as puncture tissues).
• High background and low resolution: Nonspecific antibody binding, magnetic bead adsorption and other factors will lead to a high proportion of background signals in sequencing data. It is necessary to increase the sequencing depth (usually 5-10M reads for transcription factor research and 20-40M reads for histone modification research) to improve the signal detection rate, which not only increases the cost but also makes it difficult to accurately locate the binding sites due to background interference.
• The operation is complicated and time-consuming: The whole process takes 2-5 days, involving multi-step operations such as cross-linking, crushing, and overnight IP, which requires high technical proficiency of experimenters, and the more steps, the easier it is to introduce operational errors and reduce the repeatability of the results.

The Chromatin Environment (Chawla et al., 2021)

CUT&Tag: An Innovative Scheme of Targeted In-situ Detection

To solve the above pain points of ChIP-seq, Steven Henikoff's team developed the CUT&Tag technology in 2019, which is based on the innovative design in the cross-field of molecular biology and epigenetics, and achieved the innovation of the traditional chromatin immunoprecipitation sequencing method through the following technical breakthroughs:

• Revolutionary technical strategy: The innovative strategy of "in-situ targeted cutting-direct database building" was adopted, which broke through the technical bottleneck of the traditional chromatin research process.
• Structural integrity guarantee: In-situ operation mode preserves the original state of natural three-dimensional structure of chromatin and protein-DNA interaction to the maximum extent. Nonspecific protein-DNA and protein-protein cross-linking products introduced by formaldehyde cross-linking are avoided, thus effectively reducing the experimental background noise and improving the biological authenticity and reliability of the data.
• Accurate targeted cleavage: Tn5 transposase has efficient DNA cleavage and linker ligation activity, and can specifically cleave the chromatin in the target area under the guidance of antibodies. Compared with the traditional random fragmentation method of ultrasonic fragmentation, it significantly reduces the background signal generated by non-specific cleavage, and improves the signal-to-noise ratio and analysis accuracy of sequencing data.
• Advantages of low sample demand: CUT&Tag technology shows significant advantages in low initial cell volume experiments because it reduces the sample loss caused by experimental steps.

The advent of CUT&Tag technology not only solved the long-standing technical limitations of ChIP-seq but also opened up a new paradigm of epigenetics research through innovative design of molecular mechanisms, providing an efficient and accurate technical platform for analyzing chromatin regulation mechanisms in complex biological processes.

The development of cleavage under targets and tagmentation technologies (Xiong et al., 2024)

The Core Mechanism: How CUT&Tag Works Step-by-Step

CUT&Tag technology realizes accurate protein-DNA interaction detection through four key steps, and each step is designed around specificity, low damage, and high efficiency. The specific process is as follows:

• A. Cell Permeability: Construction of antibody channels preserving nuclear structure
  • a) Core objective: Under the premise of not destroying the integrity of the nucleus, antibodies, and subsequent reagents can enter the nucleus and combine with the target protein.
  • b) Operation details: Mix the cell or nuclear sample (living cell, fixed cell or tissue sample) with the permeable buffer (such as buffer containing 0.1% Triton X-100) and incubate on ice for 5-10 minutes. Triton X-100 can gently destroy the lipid bilayer of cell membrane, but it will not disintegrate the nuclear membrane structure, ensure that chromatin remains in a natural state, and provide a channel for antibodies to enter the nucleus.
  • c) Key significance: Compared with ChIP-seq's "cell lysis-chromatin release" strategy, the permeabilization operation reduces the non-specific degradation of chromatin during the exposure process, reduces the sample loss, and lays the foundation for the low cell volume experiment.
• B. Target-specific binding: antibody-mediated precise localization
  • a) Core objective: To identify and bind the target protein or apparent modification site in the nucleus by a specific antibody, to provide an anchor point for the subsequent recruitment of transposase.
  • b) Operation details: Add the primary antibody against the target apparent marker to the permeabilized sample, and incubate at 4℃ for 1-2 hours. The antigen-binding domain of the primary antibody can accurately recognize the target protein, and its Fc segment is a reserved site for the subsequent binding of Protein A/G (pA/pG). If the affinity of the primary antibody is low, the secondary antibody can be added for signal amplification, to further improve the recruitment efficiency of transposase.
  • c) Key significance: the specificity of antibody directly determines the detection accuracy, so it is necessary to choose the proven CUT&Tag specific antibody (avoid using the antibody only suitable for ChIP, and some ChIP antibodies may not be effectively combined in the in-situ environment due to conformational problems).
• C. PA-Tn5 transposase Recruitment: Tool Assembly for Targeted Cutting
  • a) Core objective: Targeted recruitment of Tn5 transposase with the function of cleavage and linker insertion into the target protein binding region.
  • b) Operation details: Add pA-Tn5 fusion protein (Protein A/G and Tn5 transposase are connected by a flexible peptide) to the sample, and incubate at room temperature for 30 minutes. PA/G can specifically bind to the Fc segment of primary antibody (or secondary antibody), and draw Tn5 transposase to the vicinity of the target protein to form a primary antibody-secondary antibody-pA-Tn5 complex, thus realizing the targeted location of transposase. At this time, Tn5 transposase is inactive to avoid nonspecific cleavage.
  • c) Key significance: This step is the core of CUT&Tag specificity, and transposase is only enriched in the region where the target protein binds, to reduce nonspecific cleavage events from the source and lay the foundation for a high signal-to-noise ratio.
• D. Tagging: The integration of cutting and database building
  • a) Core goal: activate Tn5 transposase, cut DNA in the target area, and insert a sequencing adapter to complete the simultaneous operation of cutting and database building.
  • b) Operation details: Add activation buffer containing Mg (Mg is an essential coenzyme for Tn5 transposase) to the sample and incubate at 37℃ for 1 hour. The activated Tn5 transposase will perform double-stranded cleavage on the DNA near the binding site of the target protein, and at the same time, the sequencing Adapter preloaded on the transposase will be inserted at both ends of the cleavage site. After cutting is completed, the Tn5 transposase is inactivated by heating (such as incubating at 65℃ for 10 minutes) to terminate the reaction; Then the supernatant is collected, and the DNA fragment with sequencing linker can be obtained without additional linker connection step.
  • c) Key significance: The traditional ChIP-seq needs separate DNA purification-linker ligation-library amplification, which is cumbersome and easy to lead to sample loss. The simultaneous completion of "cutting-joint insertion" of CUT&Tag not only simplifies the process, but also reduces the sample loss in the intermediate step, which is especially suitable for experiments with low initial cell volume.

CUT&Tag-direct produces high-quality datasets on the benchtop and at home (Henikoff et al., 2020)

Key Advantages Over Traditional Methods

Compared with traditional technologies such as ChIP-seq, CUT&Tag has obvious advantages in terms of sample requirements, signal quality, operational efficiency, etc. These advantages enable it to cover research scenarios that are difficult to reach by traditional technologies:

Low Cell Input: Breaking through in Rare Sample

• Core performance: CUT&Tag can efficiently process samples of 100-1000 cells, and even achieve single-cell level detection (scCUT&Tag), while ChIP-seq usually needs 100,000 to millions of cells.
• Technical support: Permeability operation reduces sample loss during cell lysis; Targeted recruitment of pA-Tn5 reduces nonspecific cleavage, improves the enrichment efficiency of target DNA fragments, and a small number of cells can generate enough sequencing signals.
• Application value: It is possible to study the rare cell population.

High SNR: Improving Data Quality and Positioning Accuracy

• Core performance: The background signal of CUT&Tag sequencing data is extremely low, the signal Peak is sharper, and the positioning accuracy of the target site can reach the base level. However, ChIP-seq has high background signal, wide and fuzzy signal peak due to non-specific cross-linking and ultrasonic fragmentation.
• Technical support:
• Non-crosslinking operation: Avoid nonspecific protein-DNA binding caused by formaldehyde and reduce background noise.
• Targeted cleavage: Tn5 transposase cleaves only in the target protein binding region, and the DNA in non-target region is rarely cleaved, and the target fragment accounts for a high proportion in sequencing data.
• Low sequencing depth requirement: Due to the low background, CUT&Tag only needs 2-5M reads to obtain reliable results, while ChIP-seq needs 5-40M reads, which not only reduces the sequencing cost, but also reduces the calculation amount of data analysis.

Quick and Simple: Shorten Period and Lower Threshold

• Core performance: The complete process of CUT&Tag can be completed within one day (from sample processing to library construction), while ChIP-seq takes 2-5 days. The operation steps are only four core links, and there is no need for complicated operations such as ultrasonic crushing and overnight IP.
• Technical support:
• Simplified process: Cross-linking, ultrasonic crushing, cross-linking and other steps are omitted, and the operation time is reduced.
• Synchronous library building: Tagmentation step realizes the integration of "cutting-joint insertion" without a separate library building step.
• Automation compatibility: a few steps and mild operation, which can adapt to an automatic liquid treatment system, reduce human error, and improve the repeatability of results.

High repeatability: Ensure the Reliability and Comparability

• Core performance: the correlation coefficient (R) between biological repetitions of CUT&Tag is usually greater than 0.95, while the correlation coefficient between repetitions of ChIP-seq is often lower than 0.9 because of many operation steps and a great difference in sample loss.
• Technical support:
• The enzymatic reaction is controllable: The cutting efficiency of Tn5 transposase is strictly controlled by conditions such as Mg²⁺ concentration and temperature, and the reaction repeatability is high.
• Low operation error: There are few steps and no complicated operation (such as power and time optimization of ultrasonic crushing), and the results of different experimenters or laboratories are small.

Schematic of scCuT&Tag applied to cultured cells and tissues in different species (Fu et al., 2023)

Applications Unlocked by the CUT&Tag Technique

With the advantages of low sample requirements, high signal-to-noise ratio, rapidity, and simplicity, CUT&Tag technology has been widely used in many research fields, especially in scenes that are difficult to cover with traditional technologies.

• A. Dynamic study on transcription factors during development
  • a) In the process of cell differentiation or embryonic development, the binding sites of transcription factors will change dynamically, which requires high-resolution and high-efficiency technology to track this change. The rapid detection and high positioning accuracy of CUT&Tag can meet the needs, capture the dynamic process of transcription factor binding, and analyze the epigenetic regulatory network in the development process.
• B. Multiomics integration study: joint analysis of appearance and transcription
  • a) Multiomics integration (such as joint analysis of epigenome and transcriptome) can reveal the association mechanism of "epigenome-gene expression", but traditional techniques need to deal with a large number of samples separately, so it is difficult to ensure that the two sets of data come from the same cell population. However, the low sample requirement of CUT&Tag can realize "simultaneous CUT&Tag and RNA-seq in the same batch of small cells", which reduces the interference of sample heterogeneity on the association analysis of multiomics data and improves the accuracy of the analysis of regulatory mechanism.
• C. Single-cell epigenetic research
  • a) Cells in tissues are heterogeneous, so bulk CUT&Tag can't distinguish the epigenetic differences of individual cells, while scCUT&Tag can detect protein-DNA interaction at single cell level and reveal the epigenetic characteristics of cell subsets.
  • b) On the basis of traditional CUT&Tag, single cells are separated by microfluidic chip or nanopore technology, and each cell is added with a unique Cell Barcode. In the subsequent sequencing, DNA fragments of different cells can be distinguished by barcode, to realize single-cell level mapping and analyze the epigenetic mechanism behind cell heterogeneity, which provides a new perspective for tumor immunotherapy, cell typing, and other research.

Effect of the 3D structure of chromatin on the tumor immune microenvironment (Li et al., 2024)

Conclusion

With the design of "in-situ targeted cutting-synchronous database building", CUT&Tag technology has broken through the limitations of ChIP-seq in terms of samples, signals and efficiency, and has become the key technology in the study of protein-DNA interaction in epigenetics. Its characteristics of low cell input, high signal-to-noise ratio, simple operation and high repeatability play an important role in rare sample analysis, dynamic process tracking and other scenarios, and provide new means for clinical and developmental biology fields.

However, there is still room for improvement in this technology:

• Antibody compatibility is insufficient, and some low-abundance transcription factors lack special antibodies, so it is necessary to develop high-affinity and specific antibodies.
• It is difficult to process tissue samples, and the methods of dissociation and nuclear extraction need to be optimized for solid tissue permeability and cell separation.
• The ability of multi-target synchronous detection is limited, and the simultaneous detection of multiple apparent markers can be realized by multi-labeling strategy in the future.

With the development of technology, CUT&Tag is expected to be integrated with single-cell sequencing and other technologies to achieve multi-dimensional analysis, bringing new prospects for epigenetics.

References

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3. Henikoff S, Henikoff JG, Kaya-Okur HS, Ahmad K. "Efficient chromatin accessibility mapping in situ by nucleosome-tethered tagmentation." Elife. 2020 9: e63274.
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