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

内分泌干扰化学品（EDC）诱导的DNA甲基化的跨代改变，也称为 表型突变，已经在哺乳动物和鱼类中得到证实。目前还不清楚体细胞是否 可以从生殖系遗传这种跨代表位突变，以及表位突变是否可以被纠正 通过CRISPR-dCas 9表观基因组编辑工具在体内进行。鱼是开发这种工具的极好模型， 原因- a）已经在鱼类模型中证明了EDC诱导的表型和表位突变，以及2） CRISPR-Cas9基因组编辑工具可以更有效地使用，因为它们的外部胚胎 发展和透明度。在两项独立的研究中，我们发现了跨代表型， 在第一次暴露胚胎后，第三代（F2）雄性的生育力降低20-30% 第二代（F0）的药物雌激素，17 α-乙炔基炔雌醇（EE 2，模型EDC）。这些观察结果 表明胚胎EE 2暴露改变了发育中生殖细胞的编程， 跨代雄性不育表型。暴露于EE 2的鱼类雄性生殖细胞保持全局性， 低甲基化，包括DNA甲基转移酶1（Dnmt 1）表达在F0和 F2代。因此，我们假设EE 2在F0诱导生殖细胞基因组的低甲基化 由F2代生殖细胞和索马遗传，导致转录水平的改变 网络和基因表达导致男性生殖腺的生殖障碍。因为效果是 通过雄性生殖系介导，我们建议通过以下方法鉴定EE 2诱导的雄性跨代表突变： 全基因组亚硫酸氢盐测序（WGBS），并通过 CRISPR-dCas 9基因组编辑方法。拟议的研究有两个具体目标。目标1将确定EE 2- 在F0和F2代中诱导全基因组表型突变。EE 2诱导的表位突变将在 F0原始生殖细胞和精子以及F2精子和睾丸体细胞中的WGBS。独特 F0代中存在的与F2雄性生殖障碍相关的表突变 将被选中进行基因组编辑目标2将删除表突变（DNA甲基化或去甲基化标记） 通过CRISPR-dCas 9工具在F2和F3代恢复生殖健康表型， 来自F1和F2亲本谱系的胚胎。我们将微注射可编程CRISPR-Tet 1-dCas 9或 在1-细胞阶段，将CRISPR-Dnmt 3a-dCas 9或CRISPR-Dnmt 1-dCas 9导入F2和F3合子中。结果成人 将对雄性进行生殖功能恢复试验。这项拟议的R21研究的结果将用于 开发R 01项目，旨在开发表观基因组编辑工具，以纠正跨代 在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.