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Principal Investigator: Michael-Christopher Keogh
Organization: EPICYPHER, INC.
Fiscal Year: 2020
Award: $396,808
Funding agency: National Human Genome Research Institute
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.
Terms: <Acylation><Antibodies><Antibody Specificity><Arginine><Assay><Bar Codes><Bioassay><Biologic Assays><Biological Assay><Body Tissues><Cell Body><Cell Count><Cell Nucleus><Cell Number><Cells><ChIP Sequencing><ChIP-seq><Chromatin><Cleaved cell><Clinical><DNA><Data><Data Set><Dataset><Deoxyribonucleic Acid><Development><Diagnostic><Disease><Disorder><Engineering><Epigenetic><Epigenetic Change><Epigenetic Mechanism><Epigenetic Process><G(s), alpha Subunit><G(s), α Subunit><G(s)alpha><G(s)α><G-substrate><GTP-Binding Protein alpha Subunits, Gs><GTP-Binding Protein α Subunits, Gs><Genomic DNA><Genomics><Gs alpha Family G-Protein><Gsα><Gαs><Histones><Human Pathology><Hyperactive behavior><Hyperactivity><Hyperkinesia><Hyperkinesis><Hyperkinetic Movements><L-Arginine><L-Lysine><Laboratories><Libraries><Location><Lysine><Maps><Methods><Methylation><Micrococcal Nuclease><Monitor><Motor Hyperactivity><Nucleosomes><Nucleus><Performance><Phase><Post-Translational Modification Protein/Amino Acid Biochemistry><Post-Translational Modifications><Post-Translational Protein Modification><Post-Translational Protein Processing><Posttranslational Modifications><Posttranslational Protein Processing><Preparation><Protein Modification><Proteins><Protocol><Protocols documentation><Recombinants><Regulatory Ns Protein><Reproducibility><Resolution><S-nitro-N-acetylpenicillamine><SNAP><Sampling><Savings><Services><Source><Specificity><Staphylococcal Nuclease><Stimulatory Gs G-Protein><TNase><Testing><Thermonuclease><Thermostable Nuclease><Time><Tissues><Transposase><Validation><Variant><Variation><alpha Subunit Stimulatory GTP-Binding Protein><alpha-Gs><assay development><barcode><base><bio-informatics pipeline><bioinformatics pipeline><cerebellum protein substrate for cGMP dependent protein kinase><chromatin immunoprecipitation-sequencing><cleaved><clinical applicability><clinical application><cross reactivity><developmental><drug development><epigenetic regulation><epigenomics><gDNA><improved><innovate><innovation><innovative><man><man's><novel><nuclease><pre-clinical><preclinical><protein G><tissue/cell culture><tool><α-Gs>