# Non-destructive epigenetic sequencing with DNA deaminase enzymes

> **NIH NIH R01** · UNIVERSITY OF PENNSYLVANIA · 2022 · $600,000

## Abstract

PROJECT SUMMARY
This proposal aims to establish DNA cytosine deaminase enzymes as a non-destructive alternative to bisulfite
for base-resolution mapping of cytosine modifications. Epigenetic modifications to the genome play an
important role in cellular adaptation and in specialization of various cell lineages derived from the same coding
sequence. On DNA, these epigenetic changes include modification of cytosine bases at the 5-postion of the
nucleobase. The most common modification is 5-methylcytosine (5mC), followed closely by 5-
hydroxymethylcytosine (5hmC), a product of TET enzyme-mediated oxidation of 5mC. Transformations
involved in development, pluripotency and oncogenesis entail changes in the genomic patterns of 5mC and
5hmC, making it important to have robust methods to localize these modifications. The methods most
commonly used to detect these modifications involve treatment of genomic DNA with bisulfite, as the different
cytosine modification states have a different propensity for bisulfite-induced deamination which can be
analyzed by sequencing. Chemical deamination, however, can degrade the vast majority of starting DNA. As a
result, bisulfite-based approaches constrain our ability to understand the landscapes of cytosine modifications
in many small or transient cell populations, or to study how changes are coordinated across long stretches of
genomic DNA.
In this proposal, we will develop and apply DNA deaminase-based sequencing approaches that address the
major shortcomings of bisulfite. Our methods rely upon enzymatic, rather than chemical deamination, using
APOBEC3A (A3A), a DNA deaminase from the immune system repurposed for these biotechnological
applications. In our biochemical studies, we have established that A3A potently discriminates between different
cytosine modification states, and, in foundational work leading up to this proposal, we developed APOBEC-
Coupled Epigenetic Sequencing (ACE-Seq) as a non-destructive, base resolution sequencing method for
localizing 5hmC. Building on this precedent, we propose to advance two new DNA deaminase-based
sequencing approaches that can now localize 5mC and 5hmC together, providing a surrogate for bisulfite, or to
directly detect 5mC alone through deamination, which is without precedent. We will apply these methods to
address important biological questions that are refractory to bisulfite-based approaches, specifically resolving
C, 5mC and 5hmC to decipher epigenetic heterogeneity at the single cell level, revealing how in cis changes
across loci are coordinated across long stretches of DNA, and reporting on the ‘ternary code’ of all three
modifications in a single read. Our proposal therefore aims to establish DNA deaminases as a non-destructive
and more reliable means for sequencing that can displace bisulfite and its associated limitations, and to
thereby drive the widespread adoption of DNA deaminases in epigenetic sequencing in the clinic and the lab.

## Key facts

- **NIH application ID:** 10421308
- **Project number:** 5R01HG010646-04
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** Rahul Manu Kohli
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $600,000
- **Award type:** 5
- **Project period:** 2019-09-01 → 2024-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10421308

## Citation

> US National Institutes of Health, RePORTER application 10421308, Non-destructive epigenetic sequencing with DNA deaminase enzymes (5R01HG010646-04). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10421308. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*