DnD-Seq: A Versatile Platform for High-Resolution Protein-DNA Interaction Mapping in Single Cells

Background & Unmet Need

• Over 1,600 transcription factors orchestrate gene regulation through complex DNA interactions, which are often disrupted in disease and aging
• Current bulk methods like ChIP-seq and CUT&TAG require stringent conditions that disrupt weaker protein-DNA interactions and mask cellular heterogeneity by providing only population averages
• While techniques like DamID can map protein-DNA interactions genome-wide, they cannot capture the dynamic regulatory networks at single-cell resolution needed to understand cell-type specific responses and disease mechanisms
• Unmet Need: Methods that can directly identify and map non-histone protein binding events with single-cell resolution

Technology Overview

• The Technology: A sequencing method (DnD-seq) that combines nanobody-directed targeting with controlled DNA deamination to record protein-DNA binding events at single-cell resolution
• The technology utilizes an engineered split DddA enzyme with nanobody targeting enables controlled, protein-specific DNA modification that permanently marks binding sites
• The system remains inactive until deliberately activated, ensuring specific recording of protein-DNA interactions
• PoC Data: Demonstrated high specificity with clear identification of transcription factor footprints and concordance with ChIP-seq reference data
• Demonstrated CTCF profiling in primary human CD8 T cells, revealing how IDH2 mutations alter CTCF binding patterns and chromatin organization across different cell types, validated by genotype-specific analysis

Technology Applications

• Enhanced AI/ML cell modeling through high-resolution protein-DNA interaction data
• Optimization of chromatin-targeted therapeutic development
• Improved detection of off-target effects in genetic engineering
• Advanced cellular reprogramming through precise transcription factor mapping

Technology Advantages

• Integration with diverse platforms (PTA, DLP, 10X Multiome) for comprehensive analysis
• Enables simultaneous profiling of protein binding and chromatin accessibility at single-cell resolution
• Reveals cellular heterogeneity previously masked in bulk approaches
• Expandable platform allowing incorporation of additional protein-specific nanobodies

Figure: Schematic representation of the DnD-seq split enzyme system. The engineered construct comprises an inactive DddA Nterminal domain fused to a nanobody that recognizes target antibodies, and a separate C-terminal domain. When brought together at antibody-bound transcription factor (TF) sites, the enzyme becomes active.