Genome Editing Directed By Single SviCas3 In Mammanlian Cells

There have been many epoch-making achievements in the history of life sciences,among which the answers to these five basic questions constitute the cornerstone of life sciences: the nature of the genetic(DNA),the genetic mechanism(DNA double helix and half reserved copy),genetic and functional(central dogma and genetic code),genetic engineering(engineered cells extrachromosomal DNA)and genome editing(biological engineering of chromosome DNA and create).Genome editing(gene editing for short)can be divided into self-targeted genome editing(only targeting the cell's own genome)and non-self-targeted genome editing(targeting the genome of other cells).Non-self-targeting gene editing is often referred to simply as gene editing.Gene editing has become the world's most important biotechnology because of its ability to create and change life.Based on the previous findings in our laboratory:(1)single RNA-guided SviCas3 has been developed as strictly template-based genome editing tools for mammalian cells and(2)single DNA-guided SviCas3 has been developed as strictly template-based genome editing tools for microbial cells,the goal of this study is to further explore whether single DNA-guided SviCas3 can be developed as genome editing tools for mammalian cell.To validate that the genome editing directed by single RNA-guided SviCas3 in mammalian cells is successful,we first selected CAMKMT in HEK293 T genome and lepr in NIH3T3 genome as target genes as well as tcc on CAMKMT-exon 3 and tac on lepr-exon 3 as PAMs,designed and synthesized corresponding g-DNAs and t-DNAs(UHA plus egfp plus DHA),ligated g-DNA and t-DNA to plasmid skeleton and resultantly obtained genome editing plasmids p AIO-m Cherry-hcas3-t/g-?CAMKMT::egfp and p AIO-m Cherry-hcas3-t/g-?lepr::egfp,respectively.After the genome editing plasmids were transfected into host cells of HEK293 T and NIH3T3 and the transfected cells were incubated for a few days,the genomes of potential gene-edited cells glowing green fluorescence(HEK293T-?CAMKMT::egfp and NIH3T3-?lepr::egfp)in cell culture flasks were extracted and verified through DNA gel electrophoresis and DNA sequencing analysis to the PCR products of edited sequences.Our experimental results show: the conclusion in previous work of our laboratory that the single RNA-guided SviCas3 can be developed as genome editing tools for mammalian cells is correct.Subsequently,the single DNA-guided SviCas3-based gene editing(no g-DNA was required)in HEK293 T cells is explored.We selected DROSHA and CAMKMT in HEK293 T genome as target genes,designed and synthesized corresponding t-?CAMKMT::egfp and t-?DROSHA::egfp(UHA plus egfp plus DHA),ligated the t-DNAs to plasmid skeleton and resultantly obtained genome editing vectors p AIO-m Cherry-hcas3-t-?DROSHA::egfp and p AIO-m Cherry-hcas3-t-?CAMKMT::egfp,respectively.After the genome editing plasmids were transfected into HEK293 T target cells and the transfected cells were incubated for a few days,the genomes of potential gene-edited cells glowing green fluorescence(HEK293T-?DROSHA::egfp and HEK293T-?CAMKMT::egfp)in cell culture flasks were extracted and verified through DNA gel electrophores is and DNA sequencing analysis to the PCR products of edited sequences.The results show that genome editing directed by single DNA-guided SviCas3 in mammalian cells is successful.Based on this study,the conclusions of this dissertation are summarized as follows:(i)discoverting for the first time that the single DNA-guided SviCas3 can be developed as gene editing tools for mammalian cells;(ii)demonstrating again that the single RNA-guided SviCas3 genome editing mediated by is successful in mammalian cells,and no off-target effect or indel formation was observed;(iii)and therefore hypothesizing that SviCas3 might be a restriction DNA endonuclease recognizing both D-loop and R-loop.