Construction Of Gene Knockdown Cell Lines And Mice Based On CRISPR/Cas9 Technology

CRISPR/Cas9 is a new gene editing technology discovered in recent years.CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats,CRISPR),which are regular,clustered and interval-short palindromic repeats,are multiple short repetitive sequences found in archaebacteria and bacteria.CRISPR can provide their own specific protection mechanism in order to resist the invasion of foreign DNA.After exploration,the system has been used in scientific research.Researchers only need to design corresponding sgRNA of target gene,connect it to the corresponding vector,transfer it to the object of study,and then detect the corresponding gene expression by PCR and Western Blotting.The CRISPR/Cas9 system is widely used in all fields of biology and medicine due to its simpler operation,higher editing efficiency,lower cost and shorter time-consuming.This project consists of two parts.The first part is constructing MTMR14 stablyknockdown cell lines by CRISPR/Cas9 technology and subsequently analyzing its phenotypy.Firstly,we designed four pairs of sgRNAs targeting human MTMR14,ligated them to the PX458 vector,transfected it into 293 T cells,and then sequenced and detected the protein expression.We obtained the 293 T cell line with MTMR14 knockdown.After successfully constructing the CRISPR/Cas9 technology platform,we focused on lung cancer cell A549 as our research object,and then successfully constructed the MTMR14 gene knockdown A549 cell line.Next,we performed a series of phenotypic analysis of wild-type and MTMR14-knockdown A549 cells.We found that reduction of MTMR14 expression significantly promoted the proliferation of A549 cells.There was no big difference in cell cycle phase of wild-type and knock-out cells,however,the expression levels of some cell cycle regulators in these two groups of cells changed.The expression levels of CyclinD1 and CyclinE mRNA were significantly increased,while the expression leves of cell cycle negative regulators P21 and P27 mRNA were dramatically reduced.Taken together,the above results suggest that the loss of MTMR14 has a certain impact on lung cancer.The second part is establishing RhoA knockout mice based on CRISPR/Cas9 technology.We designed six pairs of sgRNAs for murine RhoA,plus the T7 promoter sequence,which allows us to transcribe them into mRNA in vitro,and then we tested in vitro the in vitro activity of these sgRNAs for better editing sgRNA.Next,these sgRNA and Cas9 mRNA were in vitro transcribed and microinjected into fertilized eggs of C57BL/6J mice.When these fertilized eggs were cultured to a two-cell stage,they were transplanted into surrogate KM mice.When the mice were born,they were genotyped and determined whether the specific gene was edited or not in these mice.In summary,on one hand,we have successfully established gene knockout cell lines by using CRISPR/Cas9 technology in our project.We also conducted a preliminary phenotypic analysis on the MTMR14 knockdown A549 cell line,found that the loss of MTMR14 have a certain impact on lung cancer.On the other hand,we have obtained sgRNAs that are highly active in editing gene knockout mice,but we have not gotten the mice whose genes have been edited yet.In the future,we will try to obtain the gene-edited mice.