Experimental Study On Gene Editing Of MiRNA-125a Using CRISPR/Cas9 System

Site-directed modification of genes is one of the important means to study gene function,and the development of gene-fixed-point modification technology has also experienced the proeess from early gene targeting to three-generation artificial endonuclease.The third-generation artificial endonuclease,Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)/CRISPR-associated(Cas),is widely used in various fields of biological research because of its simple operation,low cost and high efficiency.At present,CRISPR/Cas9 technology is mostly used in the field of gene coding,but its application in ncRNA,especially miRNA,is relatively rare.miRNAs are a class of ncRNAs with a broad biologieal activity of approximately 22 nt.Mature miRNAs regulate expression of target genes mainly by base pairing with targets on the 3' UTR of the target gene.miRNA-125a is an evolutionarily highly conserved miRNA with two biological states,5p and 3p,which have biological functions.Our previous studies showed that miRNA-125a is involved in embryo implantation and recurrent spontaneous abortion.In this study,miRNA-125a was used as an entry point to construct a fixed-point editing cell line and mouse model against miRNA-125a using CRISPR/Cas9 technology,and to explore the function of miRNA-125a in vivo and in vitro,which were mainly divided into the following four parts:First,genomic fragment knockout of pre-miRNA-125a was achieved in human chorionic trophoblast cells HTR8-S/Vneo using CRISPR/Cas9 system.Firstly,the pX458 expression vector was transformed,a set of promoter and sgRNA backbone sequences were introduced,sgRNA was designed and tested for activity;HTR8 cells were transfected with higher activity vector,monoclonal culture was performed after flow sorting,and positive monoclonal clones were screened by T7EI.The positively identified monoclonal cell lines were sequence verified to confirm changes in genomic DNA;cell function experiments were performed to explore the function of miRNA-125a at the cellular level.The results showed that the expression levels of miRNA-125a-5p and 3p in positive monoclonal cell lines were significantly lower than those in wild type,and the knockout effect was better than single-point knockout;SPACA6 gene expression was significantly up-regulated;cell function test results showed The proliferation,migration and tube-forming ability of the miRNA-125a knockdown HTR8-S/Vneo cell line were enhanced.Second,the genome-specific knock-in of pre-miRNA-125a was achieved in human chorionic trophoblast cells HTR8-S/Vneo using CRISPR/Cas9 system.Select the appropriate knock-in region in the genome,design the sgRNA and detect the activity;select the sgRNA with higher activity,and amplify the homologous arm of about 1 kb upstream and downstream of the cleavage site.Synthesize the promoter and pre-miRNA-125a sequence in vitro,then ligated to the left and right homology anns to construct the donor vector,and the HTR8 cells were co-transfected with the CRISPR expression vector,and then subjected to monoclonal eulture by flow sorting,and positive clones were identified by PCR amplification.The positively identified monoclonal cell lines were sequence verified and subjected to cell function experiments.The results showed that the expression levels of miRNA-125a-5p and 3p in positive monoclonal cell lines were significantly higher than those in wild type.The results of cell function experiments showed that the proliferation,migration and tube-forming ability of HTR8-S/Vneo cell line overexpressing miRNA-125a were inhibited.Third,genomic fragment knockout of pre-miRNA-125a was achieved in C57BL/6J mice using the CRISPR7Cas9 system.A pair of sgRNAs for fragment knockout were designed in the mouse genome,two sgRNAs were transcribed in vitro,and C57BL/6J mouse fertilized eggs were co-injected with Cas9 nuclease and transferred into maternal development to become primary(FO)mice.FO mice were identified and purified to obtain homozygous individuals with fragment knockout.Homozygous individuals take reproductive organs for knockout efficiency test and tissue section staining,and explore the effects of miRNA-125a on reproductive organs and germ cells at the tissue level.At the same time,mifepristone is used to construct early embryo-sustained mouse models at the individual level.Explore the effects of miRNA-125a on early embryonic arrest.The results showed that the expression levels of miRNA-125a-5p and 3p in the positive mice were significantly lower than those in the wild type,and the expression level of Spaca6 gene was also up-regulated.The results at the tissue level showed that the morphological structure of ovarian,uterus and testis was not knocked out by miRNA-125a.Significant effects were produced,but the contents of the epididymis were reduced;individual-level results showed that knocking out miRNA-125a had no significant effect on embryo implantation,but enhanced the sensitivity of pregnant mice to mifepristone-induced abortion.Fourth,genome-specific knock-in of pre-miRNA-125a was achieved in C57BL/6J mice using the CRISPR/Cas9 system.Selecting appropriate quasi-knock-in sites in the mouse genome,designing sgRNAs and testing for activity;constructing a donor vector containing upstream and downstream homology arms and pre-miRNA-125a.Co-injection of sgRNA,Cas9 nuclease and Donor DNA in vitro into C57BL/6J mouse fertilized eggs,and transferred into the mother to develop into primary(FO)mice;FO mice were identified and purified to obtain homozygous individuals with fixed-point knock-in.Homozygous individuals take the reproductive organs for knock-in efficiency test and tissue section staining,and explore the effects of miRNA-125a on reproductive organs and germ cells at the tissue level.The study found that the expression levels of miRNA-125a-5p and 3p in positive mice were significantly higher than those in wild type,and the base mutation of the knock-in sequence affected the overexpression of miRNA.Tissue-level results showed that overexpression of miRNA-125a did not significantly affect the mor:phology of the testis,but it also reduced the content of the epididymis.In summary,we successfully constructed miRNA-125a site-specifie knockout and knock-in cell lines using CRISPR7Cas9 system,and obtained homozygous C57BL/6J mice with miRNA-125a site-specific knockout and knock-in.These studies confirmed that the use of CRISPR/Cas9 system for miRNAs editing is effective and feasible.This model can continue to explore the effects of miRNA-125a on reproductive pregnancy process and molecular mechanism,which has certain practical value.