Studies On The Function Of MiR-2954 And YY1 In Chicken Testicular Sertoli Cells Regulating Spermatogenesis By CRISPR/Cas9 And Overexpression

MiRNAs play important roles in the biological processes of tissue and organ development,diseases occurrence,cell proliferation and apoptosis,and other biological process.In our previous studies,we found that miR-2954 was highly expressed in males of chicken embryonic gonads and other tissues,and it may play an important role in the regulation of chicken gonad development by targeting YY1 and other genes.However,the role of miR-2954 in the development of male gonad and its molecular regulation mechanism need to be confirmed in vivo.In order to study the function of microRNAs in chickens and determine the role of YY1 in testicular development,a lentivirus vector targeting at chicken miR-2954 was constructed by CRISPR/Cas9 technology to verify whether the system can effectively edit genes in chicken primary testicular Sertoli cells.Meanwhile,the target gene of miR-2954,YY1,how to regulate the Sertoli Cells in Chicken testis was studied by using over-expression and RNA-seq technology.The results of this study are as follows:1.Four sgRNAs were designed and screened by online software.Three different lentiviral expression vectors,including pLenti-sgRNA-eGFP,LentiCRISPRV2 GFP and Lenti-sgRNA-CAS9-mcherry,were selected to construct the CRISPR/Cas9 recombinant vector for miR-2954 sgRNAs.2.The CRISPR/Cas9 recombinant plasmids were transfected into chicken DF-1 cell line.Four sgRNAs of chicken miR-2954 were confirmed to have gene editing ability by T7E1 digestion,mutant sequencing and quantitative detection for miR-2954.3.The sgRNA3 recombinant vectors of LentiCRISPRV2 GFP and Lenti-sgRNA-CAS9-mcherry with different fluorescent proteins were packaged and the titer was detected in 293 T cells.The results showed that the viral titer of Lenti-sgRNA-CAS9-mcherry reached 2E+8TU/mL and could successfully infect primary testicular Sertoli cells of chickens.4.YY1,the target gene of miR-2954,was overexpressed in chicken testicular Sertoli cells.Quantitative PCR results showed that the expression of YY1 was significantly increased in the overexpression group compared with the control group,indicating that YY1 was successfully overexpressed in chicken testicular Sertoli cells.5.A total of 2955 differentially expressed genes were identified by transcriptome sequencing of YY1 overexpression and control group,of which 819 were up-regulated and 2136 were down-regulated.Through GO and KEGG analysis,we found that most of these genes were concentrated in the pathway for spermatogonial regulation by Sertoli cells.We also found AR and INHBA,the most important two genes involved in regulating spermatogonia in Sertoli cells,have significant differences after overexpression of YY1.This suggests that miR-2954 may regulate gonadal development by regulating YY1 to influence the secretion of cytokines and hormones in Sertoli cells to influence the generation and differentiation of male spermatogonia.