Genome-wide CRISPR/Cas9 Screening And Mechanisms Study On Genes Of Pancreatic Beta Cell Neogenesis

Type 1 diabetes（T1DM）,as one kind of insulin-dependent diabetes,is mainly manifested by the progressive loss of insulin-secretingβcells and blood glucose imbalance in clinical diagnosis.Insulin injection is the main symptomatic treatment for T1DM.In additional,some surgical treatments such as micro-island transplantation,have been became a potential therapy after thirty years development.However due to the limitation of donor pancreas,the transplantation surgery could not be widely applied.More and more studies have shown that pancreatic ductal cells are emerging into expandedβ-cells^[1,2].Though genome-wide CRISPR/Cas9 have been proved to be useful in screening the regulation genes in different type of diseases^[3-5].Therefore,here we identified the critical genes that promote the regeneration of labeled pancreatic ductal cells toβorβfunction cells based on an unbiased whole genome CRISPR/Cas9 screening system,which could increase the potential treatment strategy for T1DM.Part1.Whole-genome CRISPR/Cas9 screening of pancreaticβ-cell neonatal regulatory genesBy constructing a genome-wide CRISPR screening platform,we selected key regulatory genes for pancreatic ductal cells（PANC-1）to regenerate intoβcells.REPB lentivirus and GeCKO v2 library lentivirus were transfected into PANC-1 cells,RIP3.1-GFP-expressing cell population was obtained by antibiotic screening,negative control cells were treated without antibiotics;Flow cytometric sorting was used to screen the high GFP fluorescence intensity cells.GFP positive cells and negative control cells were collected and sequenced by deeply NGS.Relying on the constructed REPB reporting component,complete genome-wide CRISPR screening of neonatal regulatory genes from ductal cells toβcells was detected.The genes included ITPKA,THAP8,OUTB1,ALDH3B2,RUNX3,GPI,PRR12,RASA3,TMEM2,MBOAT7,KIF27 and SNX29,etc.twelve genes,were identified to be the potential regulating genes for regenerating of pancreatic ductal cells intoβcells.The preliminary evidence showed that the ALDH3B2 gene may have a critical role in the regulation ofβcell neogenesis by DNA sequencing,q PCR,and immunofluorescence staining.Part 2 Study on the hypoglycemic effect of ALDH3B2 gene modified PANC-1 cellsFirstly,the role of ALDH3B2 gene in the regulation ofβ-cell regeneration was observed by constructing the ALDH3B2 gene knockout PANC-1 cell line（ALDH3B2-KD）.The expression level of ALDH3B2 protein was verified by Western blot,q PCR was used to detect the effect of ALDH3B2 gene in pancreaticβ-cell transcription,endocrine hormones genes,insulin secretion genes and pancreatic duct marker-related gene expression level for PANC-1 cells;IF staining was used to detect the expression levels of insulin and C-peptide.Next,glucose secretion stimulated insulin secretion（GSIS）experiment and transmission electron microscopy were performed to study the insulin secretion of ALDH3B2-KD cells in vitro.The diabetic mice were used to observe the effect of ALDH3B2-KD cells transplantation in hypoglycemic function.The data showed that knocked out ALDH3B2 gene could significantly down-regulate ALDH3B2 protein expression in PANC-1 cells;ALDH3B2-KD cells significantly up-regulate m RNA levels ofβ-cell-related genes,whileβ-cell specific insulin and C-peptide protein expression increase.The number of insulin-secreting vesicles of ALDH3B2-KD cells were significantly increase;In one hand,the blood glucose showed significant decrease post of ALDH3B2-KD cells implant in vivo,in another hand the glucose tolerance level of diabetic mice significantly improved.Finally,small hairpin RNA technology was used to modify different sites to construct ALDH3B2 gene silencing cell line（sh RNA）,and the function of ALDH3B2 gene was verified again to eliminate off-target effects.Therefore,the ALDH3B2 gene plays an important role in the process of pancreatic ductal cell regeneration to beta cells.The higher efficiency of ALDH3B2 gene interference,the higher rate of pancreatic ductal cell regeneration transfer to beta cells.Compared with the existing technology of stem cell differentiatedβ-cell transplantation,the treatment of ALDH3B2-KD cells is easier to achieve clinical practical application and may develop into an innovative direction for the treatment of T1DM in the future.Part3.Study on mechanism and application in ALDH3B2 gene therapy in T1DM Previous studies have shown that DNA methylation is an important determinant of gene expression,and the insulin gene promoter is less methylated in pancreatic beta cells^[6].To indicate the possible mechanisms of ALDH3B2 gene regulating pancreatic ductal cells into beta cells,we used DNA methylation high-throughput sequencing to do a preliminary research.The data showed that ALDH3B2 gene knockout can significantly reduce the level of demethylation of the insulin promoter in PANC-1cells,which suggested that ALDH3B2 gene regulate the pancreatic ductal cells differentiate into beta cells by reducing DNA methylation levels.Secondly,since ALDH3B2 protein belongs to the Aldehyde dehydrogenase（ALDH）family,we use ALDH enzyme inhibitors:N,N-diethylaminobenzaldehyde（DEAB）and disulfiram（DSF）,the results confirmed that ALDH enzyme inhibitors could reduce the expression of ALDH3B2 protein in human ductal cells and up-regulate the m RNA level of insulin,suggested that specific inhibitors of ALDH3B2 could provide new strategies for the clinical treatment of T1DM.Finally,we used microbeads to separate and purify primary mouse and human pancreatic duct cells.By using CRISPR/Cas9ALDH3B2 gene editing technology,the data showed that mouse and human primary pancreatic duct cells could differentiate intoβ-cells.The application of CRISPR/Cas9ALDH3B2 gene editing technology in human primary cells provides new possibilities for gene editing cell transplantation therapy for T1DM,also indicates a theoretical foundation for ALDH3B2 gene as clinical targets.