# Epigenomic profiling of complex tissues with single-cell CUT&RUN

> **NIH NIH R01** · FRED HUTCHINSON CANCER RESEARCH CENTER · 2021 · $687,696

## Abstract

Summary
Human genetic regulatory elements remain poorly defined, in large part owing to technical
limitations of methods that have been used to map specific components of the chromatin landscape.
By far the most popular of these methods is ChIP-seq, which is used in thousands of laboratories and
is a staple of large infrastructural projects such as NIH's ENCODE and Epigenomic Roadmap
consortia. However, in 1½ years since its introduction, our novel Cleavage Under Targets & Release
Using Nuclease (CUT&RUN) antibody-tethered nuclease method has surpassed standard ChIP-seq
in efficiency and resolution by orders of magnitude. We have developed extensions to CUT&RUN
that make it more generally applicable to biological problems, and have introduced a high-
throughput automated format for research and clinical applications.
To extend the utility of CUT&RUN to heterogeneous cells and tissues, we propose to develop two
single-cell CUT&RUN strategies with distinct advantages. In both cases, we apply in situ ligation to
CUT&RUN fragments in bulk, for which we present preliminary proof-of-concept data. One strategy
uses direct barcoded amplification of CUT&RUN fragments in nanowell chip arrays and the other
uses split-pool combinatorial barcoding. To help guide technology development and to further our
understanding of important developmental pathways, we will apply single-cell CUT&RUN to
human CD34+ primary hematopoietic cells and Drosophila germline tissues. We will also develop
novel computational tools customized for CUT&RUN data that take advantage of the base-pair
precision of cleavages by using fragment length and position for peak-calling and for identification of
active genetic regulatory elements. We will use standard computational tools that have been
developed for single-cell RNA-seq data to delineate cell-type, and we will develop software for
simultaneous mapping of adjacent transcription factors, histone marks and RNA Polymerase II
within single cells to deduce enhancer-promoter-gene combinations. Finally, we will exploit the
ability of CUT&RUN to detect a new general nucleosome feature that we recently discovered in
which regulatory elements are marked by asymmetrically unwrapped nucleosomes.
Taken together, our proposal will introduce a low-cost high-throughput single-cell epigenome
characterization strategy that applies to the wide variety of basic research and clinical applications
that require information from the activity of genetic regulatory elements.

## Key facts

- **NIH application ID:** 10089227
- **Project number:** 5R01HG010492-03
- **Recipient organization:** FRED HUTCHINSON CANCER RESEARCH CENTER
- **Principal Investigator:** Steven Henikoff
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $687,696
- **Award type:** 5
- **Project period:** 2019-04-19 → 2024-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10089227

## Citation

> US National Institutes of Health, RePORTER application 10089227, Epigenomic profiling of complex tissues with single-cell CUT&RUN (5R01HG010492-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10089227. Licensed CC0.

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