The CUT&RUN assay, which stands for Cleavage Under Targets and Release Using Nuclease, is a chromatin profiling method used to map protein-DNA interactions with high specificity and low background. It is widely applied to study transcription factors, histone modifications, and chromatin-associated proteins in a more streamlined way than traditional chromatin immunoprecipitation. Because it requires less starting material and produces cleaner signals, it has become an important tool in epigenetics and gene regulation research.

Principle of the Method

CUT&RUN works by binding a target-specific antibody to a chromatin protein in permeabilized cells or nuclei, followed by recruitment of a protein A- or protein G-fused micrococcal nuclease. Once activated, the nuclease cleaves DNA near the antibody-bound protein, releasing small DNA fragments that can be purified and analyzed. This targeted cleavage strategy reduces background noise because only DNA near the protein of interest is liberated.

Main Experimental Steps

The assay generally begins with permeabilized cells or nuclei that are incubated with a highly specific antibody against the protein of interest. After binding, the nuclease fusion protein is added, and controlled cleavage is initiated to release protein-associated DNA fragments. The purified DNA can then be analyzed by qPCR, sequencing, or other genomic methods depending on the experimental goal.

Applications in Research

CUT&RUN is commonly used to study transcription factor occupancy, histone modification profiles, and regulatory chromatin landscapes. It is especially useful in cell types or tissues where sample amounts are limited, since it works efficiently with low input material. Researchers use it in developmental biology, cancer studies, immunology, and stem cell research to identify regulatory elements and understand chromatin organization.