# Correction of endocrine disruptor-induced transgenerational epimutations by CRISPR-dCas9

> **NIH NIH R21** · UNIVERSITY OF NORTH CAROLINA GREENSBORO · 2020 · $218,250

## Abstract

Endocrine disrupting chemical (EDC)-induced transgenerational alterations in DNA methylation, also called
epimutations, have been demonstrated in mammals and fish. It is not yet clearly understood if somatic cells
can inherit such transgenerational epimutations from germline and whether the epimutations can be corrected
by CRISPR-dCas9 epigenome editing tools in vivo. Fish are excellent models to develop such tools for two
reasons- a) EDC-induced phenotypes and epimutations have been demonstrated in fish models, and 2)
CRISPR-Cas9 genome editing tools can be used more efficiently because of their external embryonic
development and transparency. In two independent studies, we have found transgenerational phenotypes with
20-30% reduced fertility in the males at the third generation (F2) after exposure of embryos during the first
generation (F0) to the pharmaceutical estrogen, 17a-ethinylestradiol (EE2, a model EDC). These observations
suggest that embryonic EE2 exposure alters programming of developing germ cells leading to
transgenerational male subfertility phenotype. The male germ cells from EE2-exposed fish maintained global
hypomethylation including DNA methyltransferase 1 (Dnmt1) expression at a suppressed state at both F0 and
F2 generation. We, therefore, hypothesized that EE2 induces hypomethylation in germ cells' genome at F0
generation which is inherited by F2 generation germ cells and soma resulting in alterations of transcriptional
networks and gene expression leading to reproductive impairment in male gonads. Since the effects were
mediated by male germ line, we propose to identify EE2-induced transgenerational epimutations in males by
whole genome bisulfite sequencing (WGBS) and to correct phenotype-specific epimutations in vivo by
CRISPR-dCas9 genome editing method. The proposed study has two specific aims. Aim 1 will identify EE2-
induced genome-wide epimutations in F0 and F2 generations. EE2-induced epimutations will be analyzed in
F0 primordial germ cells and sperm and in F2 sperm and testicular somatic cells by WGBS. Unique
epimutations that were present in F0 generation and that are associated with F2 male reproductive impairment
will be selected for genome editing. Aim 2 will remove epimutations (DNA methylation or demethylation marks)
by CRISPR-dCas9 tools to recover a reproductively healthy phenotype at F2 and F3 generations using
embryos from the F1 and F2 parental lineages. We will microinject programmable CRISPR-Tet1-dCas9 or
CRISPR-Dnmt3a-dCas9 or CRISPR-Dnmt1-dCas9 into F2 and F3 zygotes at the 1-cell stage. Resulting adult
males will be tested for recovery of reproductive function. Results from this proposed R21 study will be used to
develop a R01 project directed toward development of epigenome editing tools to correct transgenerational
phenotypes in vivo in other model organisms. Results from this study provide bring new insights into epigenetic
mechanisms underlying transgenerational diseases in humans.

## Key facts

- **NIH application ID:** 9894194
- **Project number:** 1R21HD098621-01A1
- **Recipient organization:** UNIVERSITY OF NORTH CAROLINA GREENSBORO
- **Principal Investigator:** Ramji Kumar Bhandari
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $218,250
- **Award type:** 1
- **Project period:** 2019-12-15 → 2021-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9894194

## Citation

> US National Institutes of Health, RePORTER application 9894194, Correction of endocrine disruptor-induced transgenerational epimutations by CRISPR-dCas9 (1R21HD098621-01A1). Retrieved via AI Analytics 2026-06-14 from https://api.ai-analytics.org/grant/nih/9894194. Licensed CC0.

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