Establishment Of CRISPR-mediated Gene Knockout And Lentivirus-mediated Gene Overexpression Systems In Chickens

With the development of reverse genetics technology,it is more conducive to the genetics molecular basis research for the growth and development of livestock and poultry,so as to provide theoretical and technical support for improving the production characteristics of livestock and poultry and accelerating the genetic breeding progress.In the process of molecular breeding,gene knockout and gene overexpression are the first steps to study the molecular regulation mechanism,and their related techniques have been gradually improved and widely used in various species.However,the development of these technologies in poultry lags behind that in mammals due to the particularity of breeding physiology of poultry represented by chicken.Therefore,in order to promote the further application of gene knockout and overexpression technology in chickens,it is necessary to establish the optimization system and explore the feasibility of the system in the gene function study.In terms of gene knockout technology,CRISPR/Cas9 system developed in recent years has improved the shortcomings of traditional knockout technology in terms of efficiency,targeting accuracy,multi-gene knockout,species limitation and cost.There were some reports using CRISPR/Cas9 system to targetedly knockout genes of chicken with some problems,such as low editing efficiency.In this study,we used paired g RNAs to target the same gene exon in chicken cells,and established a chicken gene knockout system from the strategies of sgRNA,the dual fluorescence-resistant reporter system based on repair mechanism,amplification sequencing and T7E1 detection.In terms of gene overexpression,lentivirus is regarded as an efficient method for gene overexpression due to its integration effect,efficiency and safety.It is widely used to reveal the mechanism and function of genes,but its application in chicken primary cells or embryos is not satisfactory.So this study constructed lentivirus vectors and completed high titer lentivirus packaging,successfully transduced primary chicken cells,and detected the overexpression effect of transduced functional genes.The results are as follows:1.Establishment of chicken gene knockout system based on CRISPR/Cas9 system.The single sgRNA expression vector and the resistance repoter vector based on repair mechanism were constructed by designing pairs of sgRNA targeting six target sites respectively.After co-transfection into DF-1 cell lines,positive cells were enriched by puromycin,and the editing efficiency was up to 99.65%.Meanwhile,deletion fragment size distribution analysis showed that compared with single sgRNA,double sgRNA was more likely to cause a large number of deletion fragments above 30 bp,but the insertion and deletion(indel)percentage was similar to that of single sgRNA.Secondly,by comparing the editing efficiency of the test,it can be proved that the sensitivity of chicken intracellular amplicon sequencing is much higher than that of T7E1 assay.2.Establishment of chicken gene overexpression system based on lentivirus integration system.The recombinant lentiviral vector was constructed for lentivirus packaging,and the obtained high-titer lentivirus was transduced into.The expression of target gene was detected by RT-PCR,which proved successful infection.Phenotypic analysis of cell characteristics,passage upper limit,growth curve and cell senescence demonstrated that this overexpression system could lead to the overexpression of proliferative genes in CEF,and enhance the proliferation and passage ability of CEF.In conclusion,this study established the chicken gene knockout and overexpression system.The CRISPR/ Cas9-based knockout system can improve the efficiency of chicken gene editing through enrichment and complete the knockout of large gene fragments.Lentivirus-based overexpression systems allow stable overexpression of genes in CEF through high titer lentivirus packaging.This study can provide reference for subsequent research on chicken gene knockout or overexpression,and lay a foundation for further optimization of the system.