The Effect And Mechanism Of CRISPRa Targeting NF1 Gene Promoter In The Cell Model With Haploinsufficiency

Haploinsufficiency refers to a genotype in which one allele of a gene can express normally after mutation or deletion,but the translated protein expression of the gene is only half of the normal protein expression level,which is not enough to maintain the normal physiological function of the gene,and then leads to the occurrence of many human diseases.The diseases it causes include cancer,nervous system diseases,developmental disorders,immune diseases,metabolic disorders and so on.The mutation of NF1 tumor suppressor gene can form a genotype with haploinsufficiency,which leads to type I neurofibromatosis and seriously threatens the life safety of patients.At present,the clinical treatment for this kind of disease is mainly surgery and drugs,and there is no systematic therapy.In recent years,the emergence of gene therapy has brought hope for the cure of such diseases.Our research group used CRISPR/Cas9 gene editing technology to obtain U251 cell model with haploinsufficiency,targeted NF1 gene promoter through CRISPR/d Cas9(CRISPRa)system,and up-regulated NF1 gene expression endogenously,which indicated that restoring or improving NF1 gene expression in U251 cell model with insufficient single dose could inhibit cell proliferation,growth and invasion.Methods:(1)Using CRISPR/Cas9 gene editing technology,the U251 cell model with insufficient single dose was constructed.(2)Taking NF1 gene promoter as the target,sg RNA was designed,and CRISPRa dual-vector up-regulation system in vitro was constructed.(3)The expression of NF1 m RNA in cells was detected by RT-q PCR method to verify whether CRISPRa up-regulation system played a role in vitro.(4)Western Blot was used to detect the expression of NF1 protein in cells,and to verify whether CRISPRa up-regulation system played a role in vitro.(5)CCK-8 and plate cloning method were used to detect the effect of NF1 gene overexpression on cell proliferation and cloning ability in U251 cell model with insufficient single dose.(6)Transwell method was used to detect the influence of NF1 gene overexpression on cell invasion ability in U251 cell model with insufficient single dose.(7)Using NF1 promoter as the target,according to the design rules of sg RNA,sg RNA was redesigned to construct a single vector up-regulation system of CRISPRa in vivo.Experimental results:(1)The U251 cell model with insufficient single dose was successfully constructed(2)The two-vector upregulation system of CRISPRa in vitro was successfully constructed.(3)Overexpression of NF1 gene in U251 cells with insufficient single dose can inhibit the proliferation,growth and invasion of cells,showing anti-tumor effect.(4)The single vector upregulation system of CRISPRa in vivo was successfully constructed.Conclusion:Endogenous up-regulation of NF1 gene expression can inhibit the growth,proliferation and invasion ability of U251 cell model with insufficient single dose,showing tumor inhibition effect.