Massively Parallel CRISPR-Cas9 Knockout Screening in Sheep Granulosa Cells for FSH Response Genes

文献类型： 外文期刊

第一作者： Liu, Zaixia

作者： Liu, Zaixia;Dai, Lingli;Bao, Yanchun;Gu, Mingjuan;Shi, Caixia;Zhou, Le;Ma, Fengying;Na, Risu;Zhang, Wenguang;Liu, Zaixia;Dai, Lingli;Bao, Yanchun;Gu, Mingjuan;Guo, Lili;Zhou, Le;Ma, Fengying;Na, Risu;Zhang, Wenguang;Dai, Lingli;Sun, Tianhao;Liu, Yongbin;Fu, Shaoyin;He, Xiaolong;Wang, Yu;Guo, Lili;Zhang, Wenguang

作者机构：

关键词： granulosa cells; FSH; massively parallel CRISPR-Cas9 library; dose-dependent genes

期刊名称：ANIMALS （ 影响因子：3.0； 五年影响因子：3.2 ）

ISSN： 2076-2615

年卷期： 2024 年 14 卷 6 期

页码：

收录情况： SCI

摘要： Simple Summary The development of ovarian follicles is mainly regulated by the follicle-stimulating hormone (FSH) released by the pituitary gland. FSH acts on the granulosa cell receptor FSHR to regulate oocyte development, follicular maturation, and ovulation. In this study, the optimal dose of FSH for promoting granulosa cells was explored by combining massively parallel sheep genome CRISPR-Cas9 knockout screening and transcriptome analysis. The CRISPR-Cas9 knockout libraries were designed on Chr 2, 3 and X and the sheep granulosa cell libraries were knocked out. This may be the first time that two methods have been combined to identify new FSH-responsive genes and pathways not foreseen in previous studies.Abstract Follicle-stimulating hormone (FSH) regulates ovarian follicle development through specific gene expression programs. Granulosa cells (GCs) are somatic cells surrounding the oocytes, secreting gonadotropins to regulate ovulation and promote follicular development. By analyzing the effects of different doses of FSH on the proliferation of GCs, we found that adding 10 ng/mL of FSH, as the optimal concentration, could promote the growth of GCs. Furthermore, we have successfully constructed the first CRISPR-Cas9 knockout library targeting the genes on chromosomes 2 and 3 and the X chromosomes of the sheep massively parallel coding gene, as well as an ovarian GCs knockout cell library. For the first time, we have exposed the knockout cell library to a concentration of 10 ng/mL FSH to explore the underlying mechanisms. Through this screening, we have identified 836 positive-negative screening genes that are responsive to FSH, thereby revealing the regulatory mechanisms and screening the functionality of candidate genes. Next, RNA-Seq of control (0 ng/mL), low (10 ng/mL), and high (100 ng/mL) doses of FSH revealed 1708 differentially expressed genes, and combined with 836 genes, we obtained 129 FSH dose-dependent genes with extremely significant differences. This enables us to delve deeper into investigating and identifying the mechanisms by which FSH regulates GCs. More generally, we have discovered new regulatory factors and identified reproductivity-associated major effectors. These findings provide novel research directions for further studies on sheep reproduction.

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