Establishment KDM2A Knockout Cell Lines And S100A9 Knockout Mice By CRISPR/Cas9 Genome Editing

CRISPR/Cas9 is a kind of adaptive immunosystem that exists in eubacteria and Archaebacteria,which can remove exogeneous genes to resist viral invasion.CRISPR/Cas9 was initially discovered in 1987,but its underlying mechanisms and functions were not fully understood until 2010.In 2013,CRISPR/Cas9 was developed as a tool for gene editing,and three important elements,Cas9 endonuclease,crRNA and tracrRNA could be incorporated as two.sgRNA(single strand guide RNA)could be introduced thro?gh crRNA and tracrRNA,thro?gh which process could enlarge the uses in genome editing.Due to the simplicity and high editing efficiencies,this technique has been employed in so many research fields,and of course,lowered the difficulties that researches must conquer during gene edition.Because of the advantages mentioned above,we rely on this technique to research the functions of targeted genes and study the roles that this gene might play in some diseases development.This project contains two parts,but both employed CRISPR/Cas9 technique.The first part in to examine the gene KDM2 A expression in asthmatic models,and confirmed that the expression levels were reduced with some uncertain mechanisms which we were not so aware of.Thus,we used this technique to construct KDM2 A gene knockout cell lines to study its functions,and we have achieved several results:1.By comparisons among different KDM2 A transcripts,we chose KDM2A-212 to set out,and harvested 4 sgRNAs: hk2-1,hk2-3,hk2-5 and hk2-7.2.By analyzing current CRISPR/Cas9 vectors,we chose pX458 vector as a targeting vector,and successfully assembled sgRNAs into pX458,constructing KDM2A-pX458 fusion vectors,and the sequencing results prove that the constructions were up to the expectations.3.We harvested 4 monoclonal cell lines by lipofectamine transfection and flow cytometry separation.4.By sequencing the four cell lines,we determined to use one knockouts,and by Real-time PCR?Western blotting,we found that these knockouts have lost the ability to express KDM2 A.5.We found that there exists an obvious sharp decrease in survival rates by CCK8(cell count kit 8).6.To determine the specific impacts that KDM2 A knockouts might exert in cell proliferation and apoptosis,we found that knockout cell shows lower viability and higher apoptosis rates compared to wild type by EdU test and Tunel test.7.The expression of TGF-?2 proliferation-related factors in mutant cells was down.The second part is that we constructed a S100A9 knockout mouse model by CRISPR/Cas9 system.This gene was screened in formal labwork,and it is shown to cause a significant rise in both acute and chronic asthmatic models,which might be correlated with asthma disease.Thus we decided to construct a S100A9 knockout mouse model to make further studies.We have achieved the success as below:1.We designed 6 sgRNAs by analysis and comparison among different S100A9 transcripts.2.We chose lentiCRISPR v2 as bone vector,and we harvested sgRNA in vitro transcript.3.By transcription in vitro,we transformed DNA into RNA,and the results were up to expectations.4.After micro-injection,the embryoes were cultured in vitro until blastaea status.28 blastaea were harvested,among which 5 were examined to be successful by blastaea test,and the success ratio achieved 17.85%.The results show that sgRNAs were synthesized correctly.5.20 mice were born after embryo transplant,which 2 mice were successfully edited,and the knockout efficiency is 10%.