The Role And Mechanism Of Arrb1 In T-cell Development In Mices

Objective:G6PD deficiency disease is a common single gene genetic enzyme deficiency disease,which cannot be cured at present.Mass screening is the main method to prevent the disease,and finding out a new treatment strategy is also the focus of the research in the world.CRISPR/Cas9 is a new gene editing technology with great clinical application value,which has the advantages of high safety,specificity,accuracy and reliability.At present,CRISPR/Cas9 has been developed in many laboratories for the treatment of pseudohypertrophic muscular dystrophy,thalassemia,hemophilia and other single-gene genetic disease.In this article,the enzyme activity and gene screening of children G6 PD in Chongqing were carried out to find out the most valuable mutation site for clinical research.In order to provide a new strategy for the treatment of G6 PD deficiency,the CRISPR/Cas9 technique is used for site repair.Methods : Polychromatic probe melting curve method and G6PD/6PGD quantitative ratio method were used to detect G6 PD gene mutation and enzyme activity respectively.We found out the mutation site with high mutation frequency and low G6 PD activity.For this site,two pairs of sgRNA were designed to construct the recombinant plasmid of PX458-G6PD-sgRNA.The editing efficiency of HEK293 cell line after transfection was detected by T7 endonuclease ? method in vivo and cas9 protease method in vitro.G6 PD DNA sequence,enzyme activity,mRNA and protein levels were detected in CRISPR/Cas9 knockout monoclonal cell lines by Sanger sequence,G6PD/6PGD quantitative ratio assay,fluorescence quantitative RT-PCR and Western Blot.Finally,combing CRISPR/Cas9 with single strand oligonucleotide was used to repair G6 PD deficiency by the mean of homologous recombination.Results : Compared Cas9 and sgRNA expression plasmid PX458-G6PD-sgRNA were successfully constructed by sanger sequencing.The editing efficiency of sgRNA1 and sgRNA2 by T7E1 endonuclease assay in vivo was 6.74% and 12.36% respectively,and that of sgRNA1 and sgRNA2 by Cas9 protease method was 15% and 17% in vitro.The activity of G6 PD enzyme and the level of mRNA in HEK293 monoclonal cell line with 1376 G > T site knockout of G6 PD gene were significantly decreased.After co-transfection of PX458-G6PD-sgRNA2 plasmid and ssODN,1376 G > T mutation site was repair and its G6 PD enzyme activity and mRNA level were restored to normal.Conclusion:This study conducted an epidemiological survey of G6 PD deficiency among children in Chongqing area for the first time and found that 1376 G > T locus is the most clinical significance.CRISPR/Cas9 repaired the G6 PD gene by homologous recombination,which laid a theoretical and practical foundation for finding a new therapy for hereditary hematological diseases such as G6 PD deficiency.