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

内分泌干扰化学物质(EDC)诱导的DNA甲基化的跨代改变，也称为 在哺乳动物和鱼类中已经证实了表型突变。目前还不清楚体细胞是否 是否可以从生殖系继承这种跨代表型突变以及这种表型突变是否可以纠正 用CRISPR-dCas9表观基因组编辑工具进行体内编辑。鱼是为两个人开发这样的工具的极好模型 原因-a)EDC诱导的表型和表型突变已在鱼类模型中得到证实，以及2) CRISPR-Cas9基因组编辑工具可以更有效地使用，因为它们是外部胚胎 发展和透明度。在两项独立的研究中，我们发现了跨代的表型 在第一代胚胎暴露后，第三代(F2)雄性受精率降低了20%-30% 产生(F0)的药物雌激素，17a-乙炔基雌二醇(EE2，一种EDC型号)。这些观察结果 提示胚胎EE2暴露改变了发育中生殖细胞的编程，导致 跨代男性亚生育表型。暴露于EE2的鱼的雄性生殖细胞保持全球 低甲基化包括DNA甲基转移酶1(Dnmt1)在F0和 F2代。因此，我们假设EE2在F0的生殖细胞基因组中诱导低甲基化 由F2代生殖细胞和胞体遗传的世代导致转录变化 导致男性性腺生殖障碍的网络和基因表达。因为影响是 在雄性生殖系的介导下，我们建议通过以下方法鉴定EE2诱导的男性跨代表型突变 全基因组亚硫酸氢盐测序(WGBS)和纠正体内表型特异的表型突变 CRISPR-dCas9基因组编辑方法。这项拟议的研究有两个具体目标。目标1将确定EE2- 在F0代和F2代诱导全基因组表型突变。EE2诱导的表型突变将在 WGBS检测F0原始生殖细胞和精子，F2精子和睾丸体细胞。独一无二 在F0代中存在的与F2代男性生殖障碍相关的表型突变 将被选中进行基因组编辑。AIM 2将去除表位突变(DNA甲基化或去甲基化标记) 通过CRISPR-dCas9工具恢复F2和F3代的生殖健康表型 来自F1和F2亲本谱系的胚胎。我们将显微注射可编程CRISPR-te1-dCas9或 在1细胞期将CRISPR-DNMT3A-dCas9或CRISPR-Dnmt1-dCas9导入F2和F3受精卵。由此产生的成人 男性将接受生殖功能恢复测试。这项拟议的R21研究的结果将用于 开发一个R01项目，旨在开发表观基因组编辑工具，以纠正跨代 其他模式生物体内的表型。这项研究的结果为表观遗传学提供了新的见解 人类跨代疾病的潜在机制。

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.