Establishing Lethal Gene Knockout Mice By CRISPR/Cas9 System

Background:CRISPR/Cas9 technology has the advantages of short experimental cycle and editing efficiency,and has been widely used in gene editing and gene knockout mouse model making.However,since the deletion of lethal genes can lead to the death of animals,the first filial generation with the general CRISPR/Cas9 technique usually cannot be born or die before sexual maturity due to the systemic knockout of lethal genes.Methods:in this study,we developed a method for making a lethal gene knockout mouse model that can be propagated and bred.?1?bioinformatics system was used to design and construct the lethal gene CRISPR/Cas9 system element;?2?the fertilized eggs were cultured in vitro and developed into two-cell stage for microinjection;?3?two-cell embryo transplantation was performed after microinjection to obtain the chimeric first filial generation;?4?the first filial generation was constructed to realize passaging and breeding.Gene sequencing was performed on the first filial generation and the F₁-hybrid mice,both of which were found to have knockout code mutations,and a dead whole-body knockout mouse model strain was successfully established.Conclusion:in this study,molecular cloning,microoperation,embryo transplantation,molecular sequencing,CRISPR/Cas9 and other technical methods were combined to confirm that the microinjection method of CRIPR/Cas9 technology in the two-cell phase of embryo can be used to making the animal model of whole-body knockout of lethal genes.