Engineered Tn5 Variants Enhance CUT&Tag Fidelity for Transcription Factors

K160A-engineered Tn5 improves recovery of GATA2 motif-containing sites in low-salt CUT&Tag and KCl-based DynaTag, preserving TF-specific motif architectures in mouse embryonic stem cells.

• Why it matters: TF-CUT&Tag is hindered by intrinsic Tn5 activity at accessible DNA and by high-salt washes that can destabilize TF–chromatin interactions, limiting accurate mapping.
• What they did: The authors engineered Tn5 by replacing individual positively charged channel residues with neutral amino acids, identifying K160A for GATA2 and K249A for CTCF, then profiled TF binding across conditions and differentiation.
• The result: K160A TF-CUT&Tag increased recovery across pluripotency TF binding landscapes in mouse embryonic stem cells and, in skeletal muscle differentiation, improved MyoG and Pax7 occupancy while revealing motif-enriched MyoG binding and a Pax7 promoter-to-motif shift.