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Harvard University

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Nat. Methods, Sep 11 2025, | https://doi.org/10.1038/s41592-025-02811-2 Coupling CRISPR scanning with targeted chromatin accessibility profiling using a double-stranded DNA deaminase

Heejin Roh^1,2,7, Simon P. Shen^1,2,7, Yan Hu^2,3,7, Hui Si Kwok^1,2, Allison P. Siegenfeld^1,2, Ceejay Lee^1,2, Marcanthony U. Zepeda^1,2, Chun-Jie Guo^2,4,5,6, Shelby A. Roseman^1,2, Caroline Comenho^2,3, Vijay G. Sankaran^2,4,5,6, Jason D. Buenrostro^2,3 & Brian B. Liau^1,2

¹Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA.
²Gene Regulation Observatory, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
³Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA.
⁴Division of Hematology/Oncology, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA.
⁵Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.
⁶Howard Hughes Medical Institute, Boston, MA, USA.
⁷These authors contributed equally: Heejin Roh, Simon P. Shen, Yan Hu.

Corresponding author
Correspondence to Brian B. Liau.

Abstract

Genome editing enables sequence-function profiling of endogenous cis-regulatory elements, driving understanding of their mechanisms. However, these approaches lack direct, scalable readouts of chromatin accessibility across long single-molecule chromatin fibers. Here we leverage double-stranded DNA cytidine deaminases to profile chromatin accessibility at endogenous loci of interest through targeted PCR and long-read sequencing, a method we term targeted deaminase-accessible chromatin sequencing (TDAC-seq). With high sequence coverage at targeted loci, TDAC-seq can be integrated with CRISPR perturbations to link genetic edits and their effects on chromatin accessibility on the same single chromatin fiber at single-nucleotide resolution. We employed TDAC-seq to parse CRISPR edits that activate fetal hemoglobin in human CD34⁺ hematopoietic stem and progenitor cells (HSPCs) during erythroid differentiation as well as in pooled CRISPR and base-editing screens tiling an enhancer controlling the globin locus. We further scaled the method to interrogate 947 variants in a GFI1B-linked enhancer associated with myeloproliferative neoplasm risk in a single pooled CRISPR experiment in CD34⁺ HSPCs. Together, TDAC-seq enables high-resolution sequence-function mapping of single-molecule chromatin fibers by genome editing.

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