Construction And Evaluation Of HeLa SND1 Knockout Gene Stable Strain By Using CRISPR/Cas9 Gene Editing System

Objective: SND1, also known as p100 protein or Tudor-SN protein. Initial studies found that the EB virus nuclear antigen 2(Epstein-Barr virus nuclear protein2) is the co-activator of SND1. The protein is widely distributed in various species, is highly conserved in different organisms, regulated a variety of important physiological processes of the cells. SND1 protein has the ability to regulate gene transcription and splicing of pre-m RNA processing, which plays an important role in the development of a variety of tumors. But so far, the mechanism of SND1 proteins involved in tumorigenesis and the development is unclear, which needs further study. Traditional knockdown the gene expression by si RNA can not be completely excepted. The latest third-generation gene editing system, CRISPR/Cas9 technology uses sequence-specific RNA molecules guide endonuclease Cas9 to the target nucleic acid and active the endonuclease to cut the double-stranded DNA, thereby complete genome modification and editing. However, the traditional CRISPR / Cas9 technology also exists off-target effects. The modified protein Cas9 CRISRP / Cas9 gene editing system can be transformed into specific nicking enzymes, resulting in only a single strand of DNA at a specific incision site, thereby activating homologous recombination(HR) intracellular mechanisms, but can avoid non-homologous end joining(NHEJ) caused by mutations, which greatly improve the efficiency and accuracy of gene editing. Therefore,we use the latest improvements CRISPR/ Cas9 gene editing system can completely knockout SND1 genes, while avoiding the serious off-target effects. We builded the stably SND1 knockout He La cells strains, in order to research the sensitivity of He La cells treated by 5-Fu chemotherapy drugs and the effect on the cell cycle, and lay the foundation for further study.Methods: Since the overlapping region of the first exon region and the gene coding region of human SND1 gene is too short, which is not conducive to design of specific sg RNA. So we select the second exon zone for sg RNA designing. So we selected the second exon zone to design the sg RNA. We designed a pair of sg RNAs that could specially identify the upstream and downstream of SND1 gene second exon, then constructed a recombinant eukaryotic expressional plasmid by the carrier of PX462. After enzyme digestion and sequencing verification, we co-transfected a pair of recombinant plasmids into He La cell, then added puromycin to cell culture to survive positive cell after 24 h. The surviving positive cells were cultured and expanded. Finally, Western Blot and immunofluorescence identification the monoclonal surviving cell was SND1 gene knockout cells. We further examined the antibiotics susceptibility of He La cells knockout SND1 gene by the 5-Fu treated. And we will study the impact of SND1 gene knockout on the cell cycle of He La cells by flow cytometry.Results: The sequencing result show that sg RNA correctly inserted into the PX462 recombinant plasmid, and He La cell had no SND1 protein exist with western blot and immunofluorescence assay. Furthermore, we examine the effect of the 5-Fu on the drug sensitivity of He La cells with SND1 knockout, results showed that when the gene SND1 was knocked out, He La cells increased sensitivity to chemotherapy, suggesting that intracellular SND1 may be involved in tumor cell resistance to chemotherapy occur. Further studies showed that the proportion of cells in G1 phase significantly increased, G2 and S phase cells were significantly reduced, indicating that cell cycle was suppressed when the SND1 gene was knocked out.Conclusion: We successfully exploited the improved CRISPR/Cas9 gen editing system to build a stably SND1 gene knockout He La cell lines and confirmed the results by immunohistochemistry assay; We further found that He La cells increased sensitivity to chemotherapy when the gene SND1 was knocked out, suggested that intracellular SND1 may be involved in tumor cell resistance to chemotherapy occur; Further studies showed that cell cycle was suppressed when the SND1 gene was knocked out. Finally, the research of He La cells knockout SND1 gene laid a good foundation for further studies.