Knockout Hnrnpk Gene Of C2C12 Cells By CRISPR/Cas9 Technique And Function Analysis

Nuclear heterogeneity Ribonucleoprotein K(Hnrnpk)is a multifunctional protein.It binds to RNA,DNA and proteins to regulat DNA transcription,RNA processing and translation,and so on.Hnrnpk plays a key role in axonal regeneration,spermatogenesis,erythrocyte maturation,ovarian development,and cancer development,but the mechanism of regulation is not very clear during myogenic differentiation.In this study,we used the CRISPR/Cas9 gene editing technique to obtain Hnrnpk knockout mice C2C12 cell line.It will provide a model of Hnrnpk knockout and to study the role of Hnrnpk in myoblasts.The results of this study are:1.Two pairs of gRNA were designed at exon 10 of the Hnrnpk gene using gRNA online design software.By ligating with pCAG-T7-Cas9 vector,Cas9/gRNA recombinant vector was obtained.The correctness of the recombinant vector was verified by PCR and TA cloning and sequencing.The recombinant vector was transfected into C2C12 myoblasts,and the cleavage efficiency was verified by PCR amplification and T7 EI digestion.2.The cells were screened by puromycin,and the single cell clones of Hnrnpk knockout were selected by limiting dilution method.Mutation and mutation types were determined by PCR and TA cloning.A total of 11 strains of Hnrnpk mutations were obtained.There were two types of mutations.One is missing three bases TCGs before the PAM sequence of target site 2.And the other lacks 108 bases that include partial target site 1 and target site 2.Through the analysis of mutant protein sequences and functional domains,and the first mutation only causes the deletion of one amino acid in the nonfunctional domain of the Hnrnpk protein.The second mutation caused a deletion of 36 amino acids in the KI domain of the Hnrnpk protein.Further western blot analysis also indicated that the second mutation corresponds to a corresponding decrease in the molecular weight of the Hnrnpk protein.Thus,the function of Hnrnpk was analyzed by mutant cells with 108 base deletions for the Hnrnpk knockout cell model.3.Morphological analysis of the mutant showed that compared with normal C2C12 cells,mutant cells become slender,tentacles decreased,cell secretion of impurities increased,cell culture process appears more dead cells.MTT analysis showed that the proliferation rate of the mutant was significantly lower than that of the control group(P < 0.05),and the contact inhibition period was not reached for 5days.Ki67 immunofluorescence showed that the cell viability of the mutant was significantly lower than that of the control group.4.The cell cycle was detected by flow cytometry.The results showed that the cell cycle of the mutant was blocked in the G2/M phase(P < 0.05),and the cell death led to a significant sub-G1 peak.5.The cells were induced to differentiate in the mutant group and the control group by 2% horse serum.Found that cells can not be fused,and cell death increased,can not form myotubes.Immunofluorescence assay was used to detect MyHC,and the expression of MyHC was not detected in the mutant cells.6.A total of 2520 differentially expressed genes were screened out from the control group and clone 5 by using the Illumina HiSeq 2000 sequencing platform.Among them,793 genes were up-regulated and 1727 genes were down-regulated.Differentially expressed genes GO analysis showed that in the biological process,the main enrichment in the biological regulation,metabolism,biological process regulation,stress response,single cell process.And in the cell component classification mainly in the cell,cell composition,membrane,membrane composition,cell lines and other significant enrichment.In the molecular function,the differential gene is mainly enriched in binding and catalysis.The clustering of differentially expressed genes showed that the expression of genes related to proliferation and differentiation of Hnrnpk gene knocked out significantly changed.The results of KEGG enrichment of differentially expressed genes showed that a total of 829 differentially expressed genes were enriched in 275 channels,of which 53 were significantly enriched.The main enriched pathway is immune system,signal transduction and signal.