CUTRUN

CUT&RUN, or Cleavage Under Targets and Release Using Nuclease, is a genome-wide method for mapping DNA-binding proteins and histone modifications with high resolution and low background. It profiles interactions in situ by combining an antibody against the target with a micrococcal nuclease tethered to protein A, which is activated to cleave DNA near the bound protein, releasing fragments for sequencing.

The protocol typically uses permeabilized cells or nuclei bound to Concanavalin A–coated beads. After incubation with a primary antibody, a fusion protein of protein A and micrococcal nuclease binds to the antibody. Calcium ions activate the nuclease, which digests DNA adjacent to the target, releasing small DNA fragments into the solution. The released chromatin fragments are purified and subjected to sequencing library preparation and high-throughput sequencing. The resulting data enable precise localization of binding sites at near-base-pair resolution.

Advantages include a high signal-to-noise ratio due to targeted cleavage, compatibility with small cell numbers, and reduced processing time compared with traditional chromatin immunoprecipitation sequencing (ChIP-seq). CUT&RUN can be used for transcription factors, histone marks, and chromatin-associated proteins, and is often performed under native (non-crosslinked) conditions.

Limitations include dependence on antibody quality and specificity, potential biases from MNase digestion, and the need for careful controls and data analysis. The method has inspired related approaches, such as CUT&Tag, and remains a widely used tool in epigenomics and gene regulation studies. It was introduced in 2017 by Skene and Henikoff.