CUT&RUN – a Modern Application for Epigenomic Research

CUT&RUN (Cleavage Under Targets and Release Using Nuclease) is a novel method to map genomic protein binding sites that combines Chromatin Immunoprecipitation followed by high-throughput sequencing (ChIP-seq) with Chromatin Immunocleavage (ChIC). In short, DNA fragmentation is achieved in situ in immobilized, intact cells without crosslinking, using micrococcal nuclease that is fused to Protein A and/or Protein G (pA/G-MNase). The fusion protein is directed to the desired target through binding of the Protein A/G moiety to the Fc region of an antibody bound to the target. DNA under the target is subsequently cleaved and released, and the pA/G-MNase-antibody-chromatin complex is free to diffuse out of the cell. DNA cleavage products are extracted and then processed by high-throughput sequencing.

ChIP has been the predominant technique to map epigenetic markers for the last decades. More recently, ChIP-seq allows localization of epigenetic makers and protein binding sites on a genomic scale and has become a mainstay application to study gene regulation. However, in spite of the evolution of the readout the basic method to enrich the DNA of interest has remained unchanged – including its shortcomings.

CUT&RUN introduces some major modifications in order to eliminate some of the ChIP-seq shortcomings. Samples are not fixed, as it is the case for ChIP-seq, which can lead to epitope masking. Chromatin is fragmented in a targeted manner by a directed nuclease cleavage from intact cells reversibly permeabilized with the mild, nonionic detergent digitonin. The nuclear envelope remains intact since digitonin replaces cholesterol, which is only present in the plasma membrane. In contrast, chromatin for ChIP is prepared by sonication or enzymatic treatment of whole cells leading to a substantial background due to genomic DNA even after immunoprecipitation DNA enrichment.

As a consequence of this superior selectivity for chromatin containing the desired epitope, CUT&RUN has considerably lower background and better signal-to-noise ratio than ChIP-seq. This leads to a higher sensitivity and renders genomic features visible that are undetectable using ChIP-seq. In addition, less sequencing depth is required. Transcription factor binding sites can be mapped at bp resolution with 106 reads. For abundant antigens such as H3K27me3 it is even possible to start with as few as 100 cells. Single-cell profiling using combinatorial indexing genomic analysis using CUT&RUN is possible since intact cells are being used.

In contrast to other methods for the genome-wide mapping of chromatin accessibility improving upon ChIP-seq – e.g. DNase1 footprinting, MNase-seq, or ATAC-seq – CUT&RUN maps specific antigens or chromatin structure markers. Other tethering approaches like DNA adenine methyltransferase identification (DamID) and Chromatin Endogenous Cleavage (ChEC) also allow specific chromatin fragmentation depending on the protein of interest. Expression of recombinant fusion proteins does however limit their scalability, and they are not suitable to address specific histone modifications.

Chromatin Immunocleavage (ChIC) does also rely on a Protein A-MNase fusion protein that is tethered to an antibody against the protein of interest to direct DNA cleavage. However, ChIC read-out is based on a Southern blot. Combination of ChIC on native cells or isolated nuclei immobilized on magnetic beads and high-throughput NGS gave rise to CUT&RUN.

The following list highlights the advantages of CUT&RUN:

• Performed In situ on non-fixed cells; no chromatin fragmentation necessary
• Low background and high sensitivity require low sequencing depth
• Possible with low cell numbers down to 100 cells depending on the antigen
• Simple, fast, amenable to automation
• Accurate quantition using heterologous spike-in DNA or carry-over E. coli DNA from the pA/G-MNase purification

If you’ve been thinking about conducting CUT&RUN, have a look at this informational site to learn more and request a CUT&RUN protocol.

Watch a webinar on CUT&RUN and CUT&Tag recorded on March 31, 2020.

References

1. Skene, P. J. & Henikoff, S. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. Elife 6, 1-35 (2017).
2. Skene, P. J., Henikoff, J. G. & Henikoff, S. Targeted in situ genome-wide profiling with high efficiency for low cell numbers. Nat. Protoc. 13, 1006-1019 (2018).
3. Meers, M. P., Bryson, T. D., Henikoff, J. G. & Henikoff, S. Improved CUT&RUN chromatin profiling tools. Elife 8, e46314 (2019).
4. Schmid, M., Rè Se Durussel, T. & Laemmli, U. K. Technique ChIC and ChEC: Genomic Mapping of Chromatin Proteins ble, and significant amounts are lost into the pellet dur-ing centrifugation. While ChIP is highly successful when applied to solu. Mol. Cell 16, 147-157 (2004).