Single-Cell Chromatin Mapping Assays

PROJECT SUMMARYEpiCypher is partnering with Dr. Kami Ahmad to develop scCUTANA™, a first-in-class and widely accessible single cell (SC) mapping technology for histone post-translational modifications (PTMs). SC analyses enable the study of rare cell types that are masked when analyzing heterogeneous tissues. Further, SC analysis allows investigators to reliably cluster and analyze specific cellular subpopulations in situ (i.e. without cell sorting), which will be key for (pre)clinical applications. Histone PTMs are the focus of many drug development programs and correlate with unique chromatin features (e.g. promoters, enhancers). Thus, SC analysis of histone PTMs will provide a powerful new tool to study chromatin structure and gene regulation, delivering novel insights that are not possible to discern with current SC approaches (e.g. scATAC-seq or scRNA- seq). Existing mapping assays for histone PTMs, specifically Chromatin ImmunoPrecipitation Sequencing (ChIP- seq), require thousands of cells to achieve adequate signal over noise (S/N), and thus have poor resolution for SC analysis. In contrast, our scCUTANA assay platform leverages EpiCypher’s ultra-sensitive Cleavage Under Targets and Tagmentation (CUTandTag) technology, which uses antibodies to bind PTMs in situ, and then applies a protein A-protein G fusion protein to tether hyperactive Tn5 transposase (pAG-Tn5) to these sites. Controlled activation of Tn5 results in fragmentation and ligation of sequencing adapters for paired-end sequencing. This ultra-sensitive method can generate high S/N mapping data using as few 1-3 million reads. Dr. Ahmad (with Dr. Steven Henikoff) has recently developed CUTandTag-based methods for SC chromatin mapping using nanowell array- and droplet-based systems. However, these nascent approaches require significant instrument investments, large sample inputs (rt100K cells), and/or are not readily amenable to workflow automation, limiting their widespread commercial utility and potential clinical application. The innovation of scCUTANA lies within 1) the development of a combinatorial cellular indexing approach that mitigates the need for expensive instruments / reagents and streamlines sample processing, 2) the ability to scale while requiring minimal cellular inputs, and 3) the use of DNA-barcoded recombinant nucleosomes to identify specific and ultra-efficient “SC- grade” antibodies that dramatically increase assay sensitivity and reliability. In Phase II, we will continue development of this “benchtop” SC genomic mapping technology, by scaling manufacturing of our pAG-Tn5 library (Aim 1), optimizing robust protocols using a range of cells and tissues (Aim 2), and developing automated workflows and scCUTANA beta kits for internal / external validation (Aim 3). The commercial scCUTANA product will comprise two 96-well plates, containing 96 uniquely barcoded pAG-Tn5 complexes (Plate 1), barcoded PCR oligos for library enrichment (Plate 2), and other reagents for sample processing. This disruptive assay platform will be economical for laboratories of all resources, providing immediate, wide access to SC genomic mapping of histone PTMs.