The Effects Of Transcription Factor IRF8 On B Cell Differentiation Based On CRISPR/Cas9

Background and Objectives:The cornerstone of humoral immunity,a major branch of adoptive immunity,is the formation of antibody produced by antibody secreting cells(ASC)or plasma cells(PC).B cells that receive activation signals in the presence or absence of T cells initiate a differentiation program that requires epigenetic and transcriptional reprogramming to ultimately form ASC.Reprogramming is accomplished through the interplay of transcription factors that initiate gene expression programs and epigenetic mechanisms that maintain these programs and cell fates.Interferon regulatory factors 8(IRF8),as a critical transcription factor in the development of immune cells,has been proven to promote ASC formation with other synergistic factors.However,its sole role and mechanisms remain elucidated.CRISPR/Cas9(Clustered Regularly Interspaced Short Palindromic Repeats/Caspase9),a commonly used gene editing technique,has been applied to primary cells from animals and clinical patients.It is guided by g RNAs to cut at specific site to achieve gene insertion/deletion.In this project,we firstly constructed a gene knockout mouse model with CRISPR/Cas9 technique starting with Ptprc encoding CD45.Based on this model,the impact of IRF8 on B cell differentiation could be explored.Moreover,we employed Assay for Transposase Accessible Chromatin(ATAC-Seq)and RNA sequencing(RNA-Seq)to investigate the epigenetic changes and reprogramming in IRF8 knockout B cells,providing new research directions for B cell differentiation.Methods:1.Electroporation:A20 cell line derived from Balb/c mice served as the first screening candidate to get the working g RNA which deleted Ptprc and IRF8 successfully;then,Cas9/RNA ribonuclease complex electroporation of primary B cells were used to further select working g RNA for next steps.2.Construct recombinant plasmid:we added hairpins to the ends of g RNAs and cloned them into Thy1.1 plasmid.Then,we transformed these recombinant plasmids into E.coli and got them amplified for lentiviral preparation.3.Hematopoietic stem cell(HSC)isolation and ex vivo culture:lineage negative cells were enriched with Miltenyi kit then c-Kit~+Sca-1~+double positive cells were sorted with FACS(Flow cytometry cell sorting)from Cas9Cd19Cre mice.Then,these cells were cultured ex vivo for 1 day and infected with lentivirus produced in Step 2,and finally transferred to X-ray irradiated CD45.1 mice.4.4 weeks after transplantation,blood was collected from submandibular vein to check immune system reconstitution and distinguish cells from host or donor by the expression of CD45.1 or CD45.2.5.8 weeks after transplantation,these mice were taken down for spleen isolation for immunity reconstitution check and distinguish cells from host or donor by the expression of CD45.1 or CD45.2.According to the expression of Thy1.1,B cells were separated into two groups and the expression of CD45 and IRF8 were compared.6.Thy1.1 positive and Thy1.1 negative B cells and CD138~+ASC were sorted by FACS for ATAC-Seq and RNA-Seq.7.Thy1.1 positive and Thy1.1 negative B cells and CD138~+ASC were sorted by FACS,and DNA was extracted for PCR(Polymerase Chain Reaction)then sent for sequencing.Sequencing results were further analyzed by TIDE(Tracking of Indels by DEcomposition)assay to quantify the efficiency of CRISPR/Cas9 gene editing.Results:1.In A20 cell line electroporation experiments,g RNA3 and g RNA5targeted Ptprc could delete CD45 and B220 efficiently,and the efficiency of g RNA1-5 targeted IRF8 deletion was over 80%.In primary B cell electroporation,g RNA3 targeted Ptprc achieved the highest efficiency.g RNA1 and g RNA2 targeted IRF8 were selected for next steps.2.HSC infected with lentivirus containing Ptprc g RNA3 could reconstitute the immune system of mice irradiated with X-ray.B220expression was significantly decreased in Thy1.1~+cells compared with Thy1.1~–cells.However,CD45.2 expression was not affected.3.HSC infected with lentivirus containing IRF8 g RNA1 and g RNA2could reconstitute the immune system of mice irradiated with X-ray.B cell differentiation was promoted in Thy1.1~+cells compared to Thy1.1 cells.Gated on B220~+CD138~–GL7~+,IRF8 expression was significantly reduced in Thy1.1~+cells.4.Analysis of the ATAC-Seq showed that compared with IRF8normally expressing cells,IRF8 knockout cells had several distinct DAR(Differentially accessible regions)in the process of differentiation,among which the degree of chromosomal accessibility of Acr3,Creb3l2,Il9r and Dkk3 changed significantly.They influenced B cell differentiation.5.RNA-Seq comparative analysis suggested that IRF8 knockdown had a significant effect on the transcriptome.The major differences were in Act B cells.The change in the transcription level of Dkk3 was consistent with the results in ATAC-Seq.GSEA analysis found that Myc-target and IFNαsignaling pathways played a role.6.TIDE assay showed IRF8 gene editing by CRISPR/Cas9 was 20%-90%in Thy1.1 positive cells.However,the efficiency in Thy1.1 negative cells was almost 0%.Conclusions:1.This project constructed a lentivirus containing g RNA infected HSC transplantation mouse model,giving a new clue of gene knockout animal model building.2.Knockout of IRF8 in this mouse model promoted B cell differentiating to ASC.Chromatin accessibility and transcriptional changes of IRF8 knockout cells were shown by next generation sequencing.