Engineered Tn5 Variants Enhance CUT&Tag Fidelity for Transcription Factors
Abstract
The CUT&Tag method performs exceptionally well for profiling histone modifications, but mapping sequence-specific transcription factors (TFs) is complicated by intrinsic Tn5 activity at accessible DNA, and the high-salt washes used to suppress this activity can destabilize TF-chromatin interactions. We engineered the Tn5 DNA-binding channel to attenuate target-DNA engagement by replacing individual positively charged amino acids with neutral amino acids. We identified a "Goldilocks" variant (K160A) with intermediate attenuation that improved recovery of GATA2 motif-containing sites under low-salt CUT&Tag and KCl-based DynaTag conditions. A second variant (K249A) had improved performance for profiling CTCF over less accessible sites characterized by sharply focused short-fragment enrichment. By comparing the K160A and K249A variants, this TF-CUT&Tag assay provides a physical readout of the local chromatin environment at TF binding sites. Applying K160A TF-CUT&Tag to pluripotency-associated TFs in mouse embryonic stem cells increased recovery across their genomic binding landscapes while preserving factor-specific motif architectures. During skeletal muscle differentiation, TF-CUT&Tag improved recovery of MyoG and Pax7 occupancy relative to standard CUT&Tag, revealing strongly motif-enriched MyoG binding and a shift in Pax7 binding from CpG-rich promoters in progenitors toward Pax7 motif-enriched sites during differentiation. Together, these results show that TF-CUT&Tag captures distinct modes of TF-chromatin interaction, including motif-directed binding sites and more salt-sensitive sites that may depend on lower-affinity DNA recognition or protein-protein interactions and are difficult to predict from DNA sequence alone.
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BibTeX
@misc{piper2026engineered,
title = {{Engineered Tn5 Variants Enhance CUT&Tag Fidelity for Transcription Factors}},
author = {Lola Piper and Emma Baird and Harry J. Ashbaugh and Olivia A. Pentecost and Sara M. Nowinski and Derek H. Janssens},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.27.762201},
url = {https://www.biorxiv.org/content/10.64898/2026.10.27.762201v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Engineered Tn5 Variants Enhance CUT&Tag Fidelity for Transcription Factors
AU - Lola Piper
AU - Emma Baird
AU - Harry J. Ashbaugh
AU - Olivia A. Pentecost
AU - Sara M. Nowinski
AU - Derek H. Janssens
PY - 2026
DA - 2026/10/27
PB - bioRxiv
DO - 10.64898/2026.10.27.762201
UR - https://www.biorxiv.org/content/10.64898/2026.10.27.762201v1
AB - The CUT&Tag method performs exceptionally well for profiling histone modifications, but mapping sequence-specific transcription factors (TFs) is complicated by intrinsic Tn5 activity at accessible DNA, and the high-salt washes used to suppress this activity can destabilize TF-chromatin interactions. We engineered the Tn5 DNA-binding channel to attenuate target-DNA engagement by replacing individual positively charged amino acids with neutral amino acids. We identified a "Goldilocks" variant (K160A) with intermediate attenuation that improved recovery of GATA2 motif-containing sites under low-salt CUT&Tag and KCl-based DynaTag conditions. A second variant (K249A) had improved performance for profiling CTCF over less accessible sites characterized by sharply focused short-fragment enrichment. By comparing the K160A and K249A variants, this TF-CUT&Tag assay provides a physical readout of the local chromatin environment at TF binding sites. Applying K160A TF-CUT&Tag to pluripotency-associated TFs in mouse embryonic stem cells increased recovery across their genomic binding landscapes while preserving factor-specific motif architectures. During skeletal muscle differentiation, TF-CUT&Tag improved recovery of MyoG and Pax7 occupancy relative to standard CUT&Tag, revealing strongly motif-enriched MyoG binding and a shift in Pax7 binding from CpG-rich promoters in progenitors toward Pax7 motif-enriched sites during differentiation. Together, these results show that TF-CUT&Tag captures distinct modes of TF-chromatin interaction, including motif-directed binding sites and more salt-sensitive sites that may depend on lower-affinity DNA recognition or protein-protein interactions and are difficult to predict from DNA sequence alone.
SN - 2692-8205
ER -