Application of CRISPR /DCAS9 systems for increasing theexpression of FSH receptor in human granulosa cells

FSH (follicle-stimulating hormone) is necessary for the ovary to produce oocytes and the production of steroid hormones. Follicular development is arrested when FSH is not present. The change from FSH-independent to FSH-dependent growth in granulosa cells depends on FSHR (follicle-stimulating hormone receptor). CRISPR activation (CRISPRa) is based on the ability of dCas9, a CRISPR protein variant that cannot act as an endonuclease, to connect to genes without altering the DNA. CRISPRa systems bind to DNA and recruit transcription factors to enhance gene expression. CRISPRa strategies use a variety of transcriptional activators, including fusion proteins and Cas system re engineering. SunTag, SAM, and VPR have all shown significant improvements over the initial dCas9-VP64 technique; therefore, there are several options to choose from when activating genes in various cell lines. The objective of this research has been established to determine if CRISPR dcas9 technology could be used to increase FSH receptor expression in HGRC1 cells (human granulosa cell line) and to investigate the possible effects of dCas9 application on intracellular pathways . The results have indicated that, the dcas9 system can activate FSHR, and increased FSH receptor activity affects several fundamental cellular events involving steroidogenic, life-sustaining, and cell death related signalling sub-pathways. After using the dcas9 system to activate FSHR in human granulosa cells, we have demonstrated a significant increase in the gene expression profile of FSHR, AKT, aromatase, MAPK8, MAPK1, ESR1, MTOR, p53, caspase 3. The examination of FSHR related proteins has revealed that the protein levels of FSHR, AKT, aromatase, and p-38 MAPK have also increased. v The functionality of dcas9 activated FSHR was tested using follitropin alpha (Gonal-f). It has also been shown that although the gene expression levels of FSHR, AKT, aromatase, MAPK8, p53, and caspase 3 increased on a time-dependent manner, the increment in MAPK1, ESR1 (Estrogen Receptor1) and MTOR was temporary. The results of our research led us to conclude that dcas9 technology can be used to increase FSHR at the gene and protein levels efficiently. Genetically engineered granulosa cell for increased FSHR can be used as in vitro models to investigate in vitro maturation (IVM), FSH hyper/hyposensitivity, polycystic ovary syndrome (PCOS) and cancer studies. Key words: Follicle, FSHR, Granulosa Cells, CRISPR/dcas9