Engineered Tn5 Variants Enhance CUT&Tag Fidelity for Transcription Factors

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.