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<p>Genome editing enables sequence-function profiling of endogenous <i>cis</i>-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<sup>+</sup> 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 <i>GFI1B</i>-linked enhancer associated with myeloproliferative neoplasm risk in a single pooled CRISPR experiment in CD34<sup>+</sup> HSPCs. Together, TDAC-seq enables high-resolution sequence-function mapping of single-molecule chromatin fibers by genome editing.</p>

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Coupling CRISPR scanning with targeted chromatin accessibility profiling using a double-stranded DNA deaminase

  • 期刊论文

  • 发表日期：

    2025年9月11日

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  • Heejin Roh,
  • Simon P. Shen,
  • Yan Hu,
  • Hui Si Kwok,
  • Allison P. Siegenfeld,
  • Ceejay Lee,
  • Marcanthony U. Zepeda,
  • Chun-Jie Guo,
  • Shelby A. Roseman,
  • Caroline Comenho,
  • Vijay G. Sankaran,
  • Jason D. Buenrostro,
  • Brian B. Liau

摘要

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.