High-resolution genomic mapping of ssDNA and associated proteins for Alzheimer's disease research

PROJECT SUMMARYEpiCypher is collaborating with Dr. Jessica Tyler (an expert in aging, DNA repair and epigenetics), to develop CUTandRUssNTM (Cleavage Under Targets and Release Using single-stranded Nuclease), a first-in-class single-stranded DNA (ssDNA) mapping technology for research into the early pathogenesis of and possible interventions for Alzheimer’s Disease (AD). The double-stranded conformation of genomic DNA (dsDNA) is essential to maintain genome stability. ssDNA forms during many cellular processes, including transcription and the processing of DNA lesions, and is rapidly sequestered by ssDNA binding proteins (SSBs) (e.g. RPA, RAD51 and BRCA1/BRCA2) to protect and facilitate any needed repair. AD is the most common form of neurodegeneration, with early pathogenesis / neuronal cell death due in part to the accumulation of DNA damage as a consequence of defective repair mechanisms (particularly homologous recombination [HR], which is heavily reliant on ssDNA signaling pathways). Improved methods for detecting and mapping ssDNA and SSB-ssDNA complexes that accompany DNA damage repair would greatly improve our understanding of how failure of these pathways contributes to AD, and potentially reveal novel drug targets and biomarkers. However, tools to study ssDNA-related signaling are lacking. The first innovation of our approach is the development of a novel immunotethering approach, wherein: 1) an antibody to an ssDNA-associated feature (e.g. SSB) is used to locally tether an ssDNA-specific nuclease to chromatin in permeabilized nuclei; 2) next, the nuclease is activated to selectively cleave nearby ssDNA and not dsDNA; and 3) cleaved fragments are collected and sequenced to yield a precise ssDNA target localization profile. The development of protein A/G (pAG) fused to an ssDNA- specific nuclease is a key innovation, as it enables the definitive identification of ssDNA associated with any localizing factor. A second innovation of our approach is the development of nucleosome spike-in controls containing either ssDNA or dsDNA, which will be used: 1) to confirm nuclease specificity; and 2) to enable quantitative comparisons in disease / control samples -/+ eventual drug treatment. The goals of this Phase I project are to develop the CUTandRUssN workflow (Aim 1) and demonstrate its ability to map SSB-ssDNA complexes in cells, thus enabling the novel study of ssDNA repair pathways in AD models (Aim 2). In Phase II, we will expand the CUTandRUssN platform to additional chromatin features (e.g. SSBs or histone PTMs) and their associated cellular mechanisms (e.g. transcription, R-loops, DNA replication). In addition, we will develop robust protocols for widely studied AD models and human post-mortem brains, including low cell input applications and assay automation to enable large-scale clinical studies. At the end of Phase II, we will launch a CUTandRUssN beta-kit and assay services, which will be marketed to researchers, drug developers, and clinicians to accelerate AD drug discovery.