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CUT&Tag, A Powerful Alternative Strategy for Studying Protein-DNA Interaction and Binding Sites

The development of deep learning sequencing and the drastic decrease in cost per base has led to major changes in genomics, but due to shortcomings in techniques used to map chromatin fragments to the genome, the complete potential of epigenomic profiling has delayed. Chromatin sequencing immunoprecipitation (ChIP-seq) has been an efficient technical tool for studying the linking role of histone modifications and genome transcription factors. However, ChIP-seq is greatly affected by low signals, elevated backgrounds, epitope masking due to cross-linking, and low yields demand large cell numbers creating a poor result. While the ChIP assay is still the primary method to investigate protein-chromatin interactions, alternative strategies are being developed to overcome some of the limitations of ChIP assays.

CUT&Tag and the Protocol

CUT&Tag (Cleavage Under Targets and Tagmentation) is a tool in molecular biology used by researchers to examine protein-DNA relationships and to distinguish sites in DNA binding for their target protein. CUT&Tag is focused on the key ChIP concepts, antibody-based target protein binding or histone interest alteration, but antibody incubation is immediately accompanied by the slicing of chromatin and library preparation instead of an immunoprecipitation phase. A Tn5 transposase merged with protein A is used by CUT&Tag assays to guide the enzyme on chromatin to the antibody attached to its target. To perform antibody-targeted tagmentation, the Tn5 transposase is equipped with sequencing adapters that generate the assembled pA-Tn5 adapter transposon). The CUT&Tag technique is sensitive, with certain histone alterations it has been confirmed to operate with as little as 60 cells.

A general view of ChIP-Seq, CUT&RUN, and CUT&Tag protocols

Figure 1. A general view of ChIP-Seq, CUT&RUN, and CUT&Tag protocols (Kaya-Okur, 2020)

There are 3 key steps in the CUT&Tag protocol:
(1) permeabilization of native/unfixed cells;
(2) implantation with the individual antibody;
(3) targeted tagmentation. The DNA is filtered after tagmentation and the CUT&Tag libraries are prepared for sequencing.

Key Features and Advantages

CUT&Tag is a modern approach for exploring protein-DNA relationships in molecular biology and provides some advancements over ChIP-Seq and CUT&RUN:

In one particular study carried out by Kaya-Okur et al., CUT&Tag is effective with low cell numbers and it can go as low as 60 cells in order to evaluate H3K27me3 profiles across the genome. Further explanation is because pA-Tn5 cleaves DNA directly at the binding site of the antibody and does not involve preparing chromatin and sonication processes that can contribute to sample loss. The capacity to interact with limited numbers of cells is a benefit for researchers focusing on particular cell types that are impossible to produce large numbers of, such as unusual neuronal populations, pancreatic islets, or stem cells. CUT&Tag can be mounted on native (unfixed) cells or nuclei, eliminating the need for normal ChIP workflows to patch, prepare chromatin, and sonification steps since the setup of sonication can be very complex and involves advanced equipment that can be costly. CUT&Tag is much quicker compared to ChIP, which is a multi-component operation. On magnetic beads, cells are inactivated and the whole process operates in a single channel. Chromatin shearing and sequencing library adapter insertion are used in the tagmentation stage at the same time, which is ideal for high production experiments. With CUT&Tag, less sequencing is needed, as the chromatin obtained at the final stage of the procedure is in the original area of the targeted protein binding sites. The shorter CUT&Tag separated DNA sequences indicate that it does not have the same deep sequencing criteria as ChIP-Seq. Regarding the CUT&Tag protocol, 3-5 million reads are enough to produce significant results. CUT&Tag is more versatile than ChIP-Seq and CUT&RUN to single-cell analyses, making it more suitable for researchers dealing with heterogeneous populations.


  1. Kaya-Okur HS, Janssens DH, Henikoff JG, et al. Efficient low-cost chromatin profiling with CUT&Tag. Nature Protocols. 2020, 15(10).
  2. Henikoff S, Henikoff JG, Kaya-Okur HS, et al. Efficient transcription-coupled chromatin accessibility mapping in situ. biorxiv. 2020.
  3. Kaya-Okur HS, Wu SJ, Codomo CA, et al. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nature Communication, 2019, 10(1).
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