Study On The Molecular Mechanism Of Constructing A Stable Cell Line With SOSS1 Subunit Knockout And SOSS1 In HR Repair

Objective: This study aimed to construct a stable cell line that knocked out the SOSSB1,SOSSB2,and SOSSC genes using a novel gene knockout technique,CRISPR/Cas9 n double-nickase technology,to provide an in vitro knock-out cell model for the in-depth study functions of SOSS-related protein.The specific mechanism of SOSSB1 subunit in HR in SOSSB1 complex was studied based on a stable cell line with SOSSB1 knockout.Methods: Using the University of California Genome website to determine the sequence and distribution of exon and intron of target DNA.Use MIT's CRISPR target sequence design site to perform sequence comparison screening against individual exons one by one to locate the desired target.Specific guide RNA(SgRNA)is used to screen higher sgRNA sequences and specific primers are added to the 5' end to design the primers.The forward and reverse primers are complementary,except for the two-terminal adapters.After annealing it was inserted with a PX462 carrier complementary to its end.The CRISPR/Cas9 n double-nickase technology distinguishes itself from traditional CRISPR/Cas9 by producing two pairs of closely-spaced sgRNAs.This operation increases the length of the targeting sequence and effectively reduces off-target effects.To increase the reliability of later experiments,two different sets of sgRNA sequences were designed for each gene,targeting different exons.The PX462 plasmid was digested with BbsI restriction enzyme to obtain a linearized PX462 vector.The synthesized positive and negative primers were annealed and the annealing sequence was connected with a PX462 linear vector under the action of a T4 ligase to construct a recombinant eukaryotic expression plasmid that can target the target gene for cleavage.Recombinant eukaryotic plasmids were identified by single digestion and corresponding sequencing.Each group(upstream,downstream,and two)of recombinant vectors was co-transfected into HeLa cells.The positive cells were selected using the corresponding concentrations of puromycin,and monoclonal antibodies were selected and cultured.Cells,using Western blot experiments to determine whether knockout effect,and use the same method to treat 293 T cells to verify whether it is repetitive.Using a constructed cell line,a DNA damage model is constructed by induction of a corresponding DNA damage agent,and immunofluorescence experiments,DNA damage sensitive experiments,cell cycle experiments,and interactions with other proteins are verified in HR Mechanism.Results:According to the sequence comparison of each exon of SOSSB1,SOSSB2 and SOSSC genes,multiple sets of sgRNA sequences targeting different gene target sites were designed.Each gene was screened for two groups of high scoring sgRNA sequences.The restriction enzyme digestion of the PX462 recombinant vector inserted into the sgRNA sequence,combined with DNA sequencing analysis,showed that the sgRNA sequences of each gene were inserted into the PX462 vector and successfully targeted the PX462 recombinant vector.Recombinant PX462-sgRNA--A and PX462-sgRNA-B plasmids targeting each gene were co-transfected into HeLa cells,using the same amount of PX462 empty vector and puromycin-resistant plasmid cells as controls.Using a 2 ? g/mL puromycin selection,the surviving cells were diluted into single cells and cultured,cultured into macroscopic monoclonal cell clusters,picked out,and expanded for culture and western blot.Western blot results showed that compared with the control group,no expression of SOSSB1,SOSSB2 and SOSSC protein was detected in the knockoutgroup,and the HeLa cell strain knocked out by the SOSS gene was successfully constructed.Similarly,in 293 T cells,no expression of SOSSB1,SOSSB2,and SOSSC proteins was detected in the knockout group compared to the control group,and the SOSS gene knockout 293 T cell line was successfully constructed.The cells knocked out by SOSSB1 have no effect on cell cycle,but they can affect the recruitment of RAD51 recombinase.Cell injury sensitivity experiments show that loss of SOSSB1 subunit will increase the sensitivity of cells to DNA damage,and at the same time can make ATR kinase pathway The levels of phosphorylation of CHK1 and CHK2 are reduced.Conclusion: In this study,a novel CRISPR/Ca9 n double-nickase technique was successfully used to construct HeLa and 293 T cell lines that knocked out the SOSSB1,SOSSB2,and SOSSC genes.The construction of this knock-out cell model laid the foundation for the subsequent study of SOSS gene function.basis.The SOSSB1 subunit has no effect on the cell cycle of DNA damage,but it can affect the recruitment of RAD51 recombinase and participate in the phosphorylation of downstream kinase proteins of HR.