The Establishment Of An Adeno-associated Virus-mediated Gene-specific Knockout System Of Testis Tissue In Vivo

Spermatogenesis refers to the process of dividing a spermatozoon stem cell into a haploid round sperm cell through a series of mitosis and meiosis,which undergo complex morphological remodeling and eventually differentiate into mature sperm.In this complex process,cells are subject to the precise regulation of many genes,and the study and analysis of this sophisticated regulatory network has become an important step in solving the mystery of spermatogenesis.However,due to the high complexity of the spermatogenesis process and the high dependence on the microenvironment in the body,there is no better culture systen to achieve a complete spermatogenesis process in vitro.Therefore,exploring new methods of spermatogenesis research is an urgent problem to be solved.In this study,the sgRNA functional element in the CRISPR Cas9 systel was inserted into the AAV expression vector by homologous recombination technology to obtain the engineered AAV-sgRNA vector;then,the human Weel 2#sgRNA was constructed into the vector and packaged in parallel with the virus;Following infection with Hela-spCas9 cells stably expressing spCas9,the assay demonstrated that the vector was stably expressed and capable of gene knockout.Further,we verified the feasibility of the above system by using the functional gene Sycp3,which is a specific expression in testis tissue and meiosis during spermatogenesis,as the target gene.First,the sgRNA target of Sycp3 was screened,and the transformed AAV-sgRNA vector was constructed and packaged in virus.Subsequently,the virus was injected into the testicular seminiferous tubule of testis tissue-specific spCas9 protein by microinjection technique,and analyzed by T7E1.The gene knockout effect of the cells in the body,and the phenotypic detection methods such as H&E staining,immunofluorescence,and spem count were used to observe the changes in spermatogenesis phenotype after knockout.As a result,it was found that the testicular tissue after knocking out sycp3 showed a significant phenotype such as volume and weight loss,germ cell shedding,and decreased mature sperm production,and most of the sperm in the testis was stagnant.This study successfully established a system for specific knockout of target genes in testis tissue in vivo,providing a new and effective means for the study of reproductive function.