Development of efficient quantitative chromatin profiling in kit and high-throughput formats - PROJECT SUMMARY Alterations in histone post-translational modifications (PTMs) are associated with diverse human pathologies. The ability to quantitatively assess these PTMs in healthy and diseased cells is essential to accelerate the development of drugs or diagnostics targeting epigenetic regulation. However, ChIP-Seq, the most widely used approach to map the genomic location of histone PTMs, is limited by poor resolution, sensitivity, and reliability. Dr. Steven Henikoff’s group recently developed CUT&RUN (Cleavage Under Targets and Released Using Nuclease) and CUT&Tag (Cleavage Under Targets and Tagmentation), powerful new ChIP- free mapping approaches with vastly improved assay performance vs. ChIP-Seq. CUT&RUN uses antibodies to locally tether protein A-, protein G-micrococcal nuclease (pAG-MNase) to chromatin in intact nuclei, followed by controlled activation of the MNase to cleave nearby DNA. Sequencing of the released DNA fragments yields precise target localization profiles using fractions of the required cellular input (100-fold less) and sequencing depth (>10-fold less) vs. ChIP-Seq. Similarly, CUT&Tag uses protein A, protein G tethered to a hyperactive transposase (pAG-Tn5), followed by controlled activation of Tn5 to deliver sequencing adaptors and paired-end amplification / sequencing directly from genomic DNA. Removing the library preparation step increases sensitivity and accelerates sample processing, providing the first tractable approach for chromatin mapping from single cells. The efficiency of these methods could now enable pre-clinical applications in a high- throughput format. However, such assays require the development of quantitative controls for antibody validation and sample normalization. Here, EpiCypher will develop QUANTUMTM, a quantitative CUT&RUN/Tag-based platform for the ultrasensitive and reliable mapping of histone PTMs. A key innovation of this proposal is the novel engineering of DNA-barcoded recombinant nucleosomes as quantitative spike-in controls for assay development, in-application antibody validation, and reliable cross-sample comparisons. In Phase I: Aim 1, we will develop a novel DNA-barcoded dNuc spike-in control panel and optimize its use for technical variation monitoring and antibody specificity testing for CUT&RUN / CUT&Tag workflows. In Phase II: Aim 2, we will apply our spike-ins to develop QUANTUM assays on various cell and tissue types (native and fixed samples) and establish methods for quantitative sample normalization using a range of sample inputs. Finally, in Aim 3 we will develop QUANTUM beta kits and automated protocols for assay services, which will be rigorously validated by EpiCypher and external laboratories. We envision QUANTUM assays will become one of the most widely used chromatin profiling tools in the epigenetics field (given the vast gain in assay metrics vs. ChIP-Seq), with the potential to open new markets for the routine and reliable analysis of limited clinical samples.
