Research On Gene Knockout Method Based On Base Conversion

Efficient and specific gene knockout technology is of great significance for the preparation of transgenic animals,the development of clinical biological therapy.Currently widely used gene knockout techniques include ZFN,TALEN,and DNA-based homologous recombination based on embryonic stem cells and these techniques will break double-strand DNA by nucleases based on the principle of mismatch repair.In practice,these techniques have obvious defects,cumbersome and inefficient,and cause a certain number of base sequence changes,which have certain influence on the coding region of the gene and the introns that may have regulatory functions on gene expression.The CRISPR/Cas9 gene editing technology developed in recent years can efficiently and quickly achieve knockout of specific gene,but its off-target effects are serious and the difficulty of control of the cutting sequence is poor.Therefore,a brand-new gene knockout technology with high efficiency and specificity is eagely needed to promote gene function research,transgenic animal preparation and biological treatment of major diseases.Recently developed base-conversion technology based on CRISPR/Cas9 gene editing can accurately convert certain single bases at the guide of gRNA,which can be achieved only by single-strand break of DNA molecules.Meanwhile,This method will not lead to deletion of the DNA base sequence.The cytosine deaminase APOBEC1 has deamination activity and converts cytosine C into thymine T.Therefore,this paper envisages that the combining cytosine deaminase and Cas9 nuclease mutant Cas9 n can realize conversion of the codon CAG,CAA,CGA on the DNA coding strand and the TGG base on the template strand into a stop codon TAG(CAG/TGG),TAA(CAA/TGG),TGA(CGA/TGG),respectively,which will interfere with the gene translation process,that is,stopping normally synthesize the protein but not affecting mRNA transcription,thereby lead to the functional knockout of specific gene but retaining possible regulatory function of RNA.In this study,five genes encoding product not less than 13 kD were randomly selected from the NCBI gene pool,namely Mus musculus aquaporin1(Aqp1),Homo sapiens transforming growth factor beta 1(Tgf?1),Mus musculus insulin II(Ins2),H(+).-transporting ATPsynthase,Homo sapiens gastrin(Gastin),to systematic analysis of the versatility of the application of this strategy.The results showed that among the five genes,multiple codons could be converted into stop codons,which evidented that the application of this strategy is extensive.Secondly,in this study,GFP gene,Dip2 a and Dip2 c genes were selected to deeply study the feasibility and efficiency of the functional knockout technology based on base-conversion strategy(including single knockout and co-knockout).The results showed that this method can significantly reduce the number of HEK293 cells stably expressing GFP and effectively conversion of coding codon to a stop codon in Dip2 a,Dip2c,Dip2 a together with Dip2 c respectively in melanoma cells B16.By DNA sequencing and RT-PCR,Western Blot,it is also found that this technology does not affect the normal transcription of the Dip2 a and Dip2 c genes,only completely blocks the synthesis of the protein,thereby achieving functional knockout and double knockout of the Dip2 a and Dip2 c genes.The incidence of single knockout are as high as 43%,and that of double knockout can reach 20%.So,the study has proved that gene knockout based on base conversion strategy is a new technology with high efficiency,high specificity and has wide applicability.The results of this thesis lay the theoretical foundation for the establishment and improvement of gene knockout methods based on base-transformation,and provide the basis for applying the novel gene knockout technology to gene function research,rapid and efficient preparation of transgenic animals and highly efficient biological treatment of major diseases.