CRISPR/DCAS9 epigenom düzenleme teknolojisi kullanılarak oositin döllenme yeteneğinin geri kazandırılması

Male and female infertility is one of the most important problems in modern world. Many studies have been conducted on environmental factors and genetic factors, which are among the causes of infertility. Melamine which is used very frequently in the industry as the raw material of many adhesives, laminated surface coatings and dishes, was shown to be one of these environmental factors. When melamine is added to dairy products, it causes a false protein increase because of its nitrogen content. Long-term exposure of the body to the chemical melamine can cause many kidney and liver problems and infertility in newborns. It was suggested to cause infertility in women by decreasing the quality of oocyte. Recently, melamine was shown to suppress the JUNO expression which was identified as the sperm IZUMO1 receptor. As JUNO is essential for fertilization, and mice with JUNO damage are infertile, effect of melamine on fertility was suggested to be related with JUNO suppression. To investigate this relationship and suggest a potential clinical approach, we aimed to establish an environmental factor-induced infertility model in oocytes and restore fertilization capacity using CRISPR/dCas9 activation systems. To this end, we first activated the JUNO gene in HEK293T cells using the CRISPR/dCas9 SAM and Tet1 systems. As shown by qPCR, a 200-fold increase was achieved in JUNO gene expression using the SAM system. Mouse sperms were shown to bind to JUNO-activated HEK293T cells, which confirmed the sperm binding capacity of the JUNO protein, and potential of our approach. To test the clinical potential of our strategy, we generated a mice model with suppressed JUNO expression consisting of three groups: control, sham, and melamine gavage. Mice were given 50 mg/kg of melamine by gavage every two days. After three weeks of treatment, significant decrease in JUNO expression was shown in melamine-exposed oocytes. Melamine was also decreased the total number of oocytes, an increase in the number of atretic follicles, and a decrease in the expression of JUNO in granulosa cells. To demonstrate the therapeutic potential of our approach, dCas9-based activator proteins and transcribed gRNA were given by microinjection into the oocytes with decreased JUNO expression. Using qPCR and IF methods, JUNO expression was demonstrated to increase back in oocytes. Using a fertilization assay, the 2-cell ratio was found to be 46% in melamine treated oocytes, while this ratio increased to 76% in melamine treated and dCas9-based activator injected oocytes. Together, our results indicated that we have generated a successful infertility model, and by utilizing CRISPR/dCas9 activation systems, we restored the fertilization capacity of oocytes. This study suggests an original approach which can be applied to overcome different infertility problems.