An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites

By P. Skene, Steven Henikoff. bioRxiv, 2016

1,423 citations recorded by the source corpus.

We describe Cleavage Under Targets and Release Using Nuclease (CUT&RUN), a chromatin profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. Unlike Chromatin Immunoprecipitation (ChIP), which fragments and solubilizes total chromatin, CUT&RUN is performed in situ, allowing for both quantitative high-resolution chromatin mapping and probing of the local chromatin environment. When applied to yeast and human nuclei, CUT&RUN yielded precise transcription factor profiles while avoiding cross-linking and solubilization issues. CUT&RUN is simple to perform and is inherently robust, with extremely low backgrounds requiring only ~1/10th the sequencing depth as ChIP, making CUT&RUN especially cost-effective for transcription factor and chromatin profiling. When used in conjunction with native ChIP-seq and applied to human CTCF, CUT&RUN mapped directional long range contacts at high resolution. We conclude that in situ mapping of protein-DNA interactions by CUT&RUN is an attractive alternative to ChIP-seq.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/602e8c1eec64c315b5302e67c7b31c8ec57f4c21

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Citations in the corpus

FactorNet: a deep learning framework for predicting cell type specific transcription factor binding from nucleotide-resolution sequential data (2017, methodology): “Given that ChIP-seq signal noise can significantly influence the accuracy of predictions (Figure 2), we propose that future data generation efforts should use protocol improvements such as ChIP-exo[57], CUT&RUN[58], or higher quality antibodies to complement the development of prediction models.”
The technological landscape and applications of single-cell multi-omics (2023, methodology): “In addition to the well-established chromatin immunoprecipitation followed by sequencing (ChIP–seq) 45 and CUT&RUN 46 methods, cleavage under targets and tagmentation (CUT&Tag), which was developed in 2019, uses Tn5 transposase to directly tagment the antibody binding site 47 .”
Sequential regulatory activity prediction across chromosomes with convolutional neural networks (2017, background): “These experiments represent 529 unique cells/tissues profiled by DNase-seq, 1136 unique cells/tissues profiled by ChIP-seq, and 595 unique cells/tissues profiled by CAGE.”
Peak calling by Sparse Enrichment Analysis for CUT&RUN chromatin profiling (2019, background): “antibody-targeted micrococcal nuclease (MNase) fusion protein to selectively digest and liberate DNA fragments at sites of protein binding while leaving the remainder of the genome behind; thus, it features exceedingly low background in comparison with ChIP-seq [10, 11].”
Targeted in situ genome-wide profiling with high efficiency for low cell numbers (2018, positive): “In contrast to standard cross-linking ChIP, in which sonication is used to fragment the chromatin to a minimum of ~200-bp fragments, exonuclease treatment in ChIP-exo or MNase digestion in high-resolution X-ChIP-seq or native ChIP approaches allows limit or near-limit digestion 6–8, 23, 39 .”