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

通过 CRISPR-dCas9 纠正内分泌干扰物诱导的跨代表突变

• 批准号: 9894194
• 负责人: Ramji Kumar Bhandari
• 金额: $ 21.83万
• 依托单位: UNIVERSITY OF NORTH CAROLINA GREENSBORO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-15 至 2021-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9894194
• 关键词: Adult; Animal Model; CRISPR/Cas technology; Cell Culture System; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Conceptus; CpG Islands; DNA; DNA Methylation; DNA Modification Methylases; DNMT3a; Development; Disease; ESR1 gene; Embryo; Embryonic Development; Endocrine Disruptors; Epigenetic Process; Estrogens; Event; Exposure to; Female; Feminization; Fertility; Fertilization; Fishes; Future Generations; Gene Expression; Generations; Genetic Transcription; Genome; Germ Cells; Germ Lines; Gonadal structure; Guide RNA; Health; Histones; Human; Impairment; In Vitro; Inherited; Invertebrates; Japanese Killifish; Mammals; Mediating; Methods; Modeling; Mus; Oral Contraceptives; Oryziinae; Parents; Partner in relationship; Paternal Exposure; Pharmacologic Substance; Phenotype; Pregnancy; Recovery; Reproduction; Reproductive Health; Seminal Vesicles; Site; Small RNA; Somatic Cell; Structure of primordial sex cell; Techniques; Testing; Therapeutic; Time; Transcription Alteration; Vertebrates; bisulfite sequencing; cost; demethylation; egg; epigenome; epigenome editing; experimental study; genome editing; genome-wide; human disease; in vivo; insight; male; male fertility; neuronal cell body; offspring; prenatal exposure; programs; promoter; pyrosequencing; reproductive; reproductive function; sex determination; sperm cell; subfertility; tool; transcriptome; transgenerational epigenetic inheritance; treatment group; virtual; whole genome; zygote

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.

内分泌干​​扰物 (EDC) 诱导的 DNA 甲基化跨代改变，也称为 表突变已在哺乳动物和鱼类中得到证实。目前尚不清楚体细胞是否 可以从种系遗传这种跨代表突变以及表突变是否可以被纠正 通过体内 CRISPR-dCas9 表观基因组编辑工具。鱼是为两个人开发此类工具的绝佳模型 原因- a) EDC 诱导的表型和表观突变已在鱼类模型中得到证实，2) CRISPR-Cas9基因组编辑工具因其外部胚胎而可以更有效地使用 发展和透明度。在两项独立研究中，我们发现了跨代表型 在第一代胚胎暴露后，第三代 (F2) 雄性的生育能力下降 20-30% 药物雌激素 17a-炔雌醇（EE2，一种 EDC 模型）的第 1 代 (F0)。这些观察 表明胚胎 EE2 暴露改变了发育中生殖细胞的编程，导致 跨代男性生育力低下表型。暴露于 EE2 的鱼类的雄性生殖细胞保持全局状态 低甲基化，包括 F0 和 F0 处 DNA 甲基转移酶 1 (Dnmt1) 表达均处于抑制状态 F2代。因此，我们假设 EE2 在 F0 时诱导生殖细胞基因组低甲基化 由 F2 代生殖细胞和体细胞遗传的一代，导致转录的改变 网络和基因表达导致男性性腺生殖障碍。由于效果是 由雄性生殖系介导，我们建议通过以下方式鉴定 EE2 诱导的雄性跨代表突变： 全基因组亚硫酸氢盐测序（WGBS）并通过以下方法纠正体内表型特异性表突变 CRISPR-dCas9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 EE2- 在 F0 和 F2 代中诱导全基因组表观突变。 EE2 诱导的表突变将在 F0 原始生殖细胞和精子，以及 F2 精子和睾丸体细胞（WGBS）。独特的 F0 代中存在且与 F2 雄性生殖障碍相关的表观突变 将被选择进行基因组编辑。目标 2 将消除表观突变（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-Tet1-dCas9 或 CRISPR-Dnmt3a-dCas9 或 CRISPR-Dnmt1-dCas9 进入 1 细胞阶段的 F2 和 F3 合子。由此产生的成人 男性将接受生殖功能恢复情况的测试。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基因 其他模型生物体内的表型。这项研究的结果为表观遗传学带来了新的见解 人类跨代疾病的潜在机制。