Preparation Of Bovine IGF2 Gene Editing Embryos By CRISPR/Cas9 Technology

CRISPR/Cas9 is a new third generation gene editing technology.Gene IGF2 encodes insulin-like growth factor 2(IGF2),which is a polypeptide promoting cell division,participating in growth and development,affecting skeletal muscle quality and fat deposition.IGF2 is highly conserved: IGF2 level,tissue growth and fat deposition of skeletal muscle will be affected,when ZBED6 binding site of IGF2 intron 3 is destroyed.The Simmental is a kind of cattle whose meat and milk production is equivalent to that of purebred cattle.However,the researches about the effect of ZBED6 binding site knockout in IGF2 intron 3 on IGF2 expression level mainly focus on mouse and pig,there is no report about the knoctout of IGF2 on cattle.Therefore,in this study,the CRISPR/Cas9 was used to edit the IGF2 gene and prepare gene editing embryo of cattle to investigate the effect of ZBED6 binding site knockout in IGF2 intron 3 on the change of IGF2 expression level.This would lay a theoretical foundation for the production of high-yield beef cattle and prepare new breeding materials for beef cattle.The main results of this study are as follows:1.Five sg RNAs were designed by CRISPR design software according to the binding site of the inhibitor bed domain containing(ZBED6)in the third intron of IGF2 and its adjacent sequence with the Miss off-target effect prediction.Futher,the corresponding PX458-sg RNA gene targeting vectors was constructed using PX458 plasmid as the skeleton.Subsequently,the editing efficiency was verified by T7E1 enzyme digestion combined with T-A cloning detection,the result showed that sg RNA1 was the most efficient with about 40% editing efficiency.2.Ten Simmental monoclonal positive cell lines with complete knockout of ZBED6 binding site of IGF2 were successfully obtained through flow cytometry combined with infinite dilution method,of which,one was homozygous mutation.3.Luciferase test and fluorescence quantitative PCR were used to analyze the effect of mutation on the expression of IGF2.The results showed that the deletion of ZBED6 binding site could enhance the activity of promoter 3 of IGF2,and improve the expression of IGF2 at the transcription level.4.The clonal embryos from #42 cell line were obtained by somatic cell nuclear transfer(SCNT),and the knockout situation of ZBED6 binding site was confirmed using T-A cloning detection.The knockout efficiency of the clonal embryos was 100%.PX458 vector skeleton residue was identified by PCR and no skeleton residue or foreign gene was detected in cell line #42 and the clonal embryo,indicating that it was bio-safety and could be used for embryo transfer.In conclusion,the CRISPR/Cas9 system constructed in this experiment could effectively edit the muscle growth related gene IGF2 of cattle,and improve the expression level of IGF2,and obtain the bovine IGF2 gene editing embryo by SCNT,which lay a foundation for molecular breeding of high-yield beef cattle.