Enhancing Resolution and Reducing Noise: How CUT&RUN and CUT&Tag Are Changing the Game

Epigenetic modifications play a critical role in gene regulation, having a profound impact on various biological processes. Chromatin immunoprecipitation (ChIP) has been the gold standard for studying epigenetic modifications and protein-DNA interactions in cells. However, traditional ChIP has its limitations, including its requirement for large amounts of starting material, long processing times, and low resolution. Recently, two novel technologies, cleavage under targets and release using nuclease (CUT&RUN) and cleavage under targets and tagmentation (CUT&Tag), have emerged as favorable alternatives to ChIP. This article will compare traditional ChIP to the newer CUT&RUN and CUT&Tag technologies and discuss their advantages and disadvantages.

During ChIP, proteins are first cross-linked to DNA. The fixed chromatin is then fragmented, and the protein of interest (e.g., transcription factor, histone modification, chromatin remodeler) is immunoprecipitated along with the associated DNA fragments. The DNA fragments can then be purified and sequenced to identify the genomic regions that are bound by the protein of interest.

While ChIP has been a valuable tool in studying protein-DNA interactions, it has several drawbacks:

• Most ChIP assays require a large amount of starting material, which can be a challenge when working with limited cell numbers or rare cell populations.
• The technique is arduous, with multiple steps involved, including cross-linking, fragmentation, and immunoprecipitation, which can take several days to complete.
• The resolution of ChIP is limited, as the immunoprecipitated DNA fragments may contain regions that are not bound by the protein of interest.

Revolutionizing Protein-DNA Interaction Studies

CUT&RUN and CUT&Tag were developed to resolve these shortcomings. These techniques, both of which are performed in situ on intact cells or nuclei without fixation, entail cleavage of chromatin at specific antibody-occupied sites by a nuclease and the subsequent, direct capture of protein-DNA complexes.

The CUT&RUN procedure involves antibody permeabilization of the nuclear membrane, followed by DNA cleavage at the antibody-bound target protein regions by a fusion protein. The fusion protein consists of a cleavage domain (such as micrococcal nuclease or MNase) and a protein A/G (pAG) domain that binds to an antibody against the protein of interest. The antibody binds specifically to the target protein, and the pAG-MNase fusion protein binds to the antibody and cleaves the chromatin at the protein-DNA interaction site, resulting in the release of the protein-DNA complex from the chromatin. CUT&Tag is similar to CUT&RUN, but the cleavage occurs in the presence of a transposase enzyme, which inserts sequencing adapters into the cleaved DNA fragments. The DNA is then amplified and sequenced.

CUT&RUN and CUT&Tag are effective technologies that have the potential to overcome some of the limitations of ChIP:

• They require less starting material. CUT&RUN can work with as little as 1000 cells, and CUT&Tag can work with less than 100 cells.
• The processing time is shorter, with CUT&RUN taking less than half a day and CUT&Tag taking less than a day to complete.
• The resolution of CUT&RUN and CUT&Tag is higher than ChIP, as the chromatin is cleaved in situ, allowing for direct capture of the protein-DNA complex.
• CUT&Tag is a single-tube reaction, which simplifies the workflow and reduces the amount of starting material required.
• Both techniques have been shown to have lower background noise than ChIP, resulting in a higher signal-to-noise ratio.

Technique Considerations

The aforementioned CUT&RUN procedure utilizes an expensive pAG-MNase fusion protein that may not always bind specifically to the protein of interest and exhibits significant A/T sequence bias, causing the target protein-interacted DNA region profiles to be seriously affected by the level of MNase digestion.

Recently, an improved CUT&RUN-Fast technique employs a novel and unique nucleic acid cleavage enzyme mix with low sequence bias to simultaneously fragment chromatin and cleave/remove any DNA sequences in both ends of the target protein/DNA complex without affecting DNA occupied by the target protein. This also greatly speeds up the process and avoids overnight incubation.

As CUT&RUN and CUT&Tag continue to be optimized and improved, they are likely to become even more widely used in the field of epigenetics. In particular, the high-resolution and low background noise of CUT&RUN and CUT&Tag make them attractive options for studying rare cell populations or low-abundance proteins. Additionally, the single-tube reaction of CUT&Tag may be particularly useful for high-throughput applications, such as single-cell sequencing.

Reference
Kaya-Okur, Hatice S et al. Efficient low-cost chromatin profiling with CUT&Tag. Nature protocols vol. 15,10 (2020): 3264-3283. doi:10.1038/s41596-020-0373-x