Gene Editing In The Mammalian Cells On The Subtype I-B-Svi CRISPR-Cas3 System

The CRISPR/Cas system is an immune system that resists the invasion of foreign nucleic acids produced by bacteria and archaea during long-term evolution.The system can cleave the target DNA by artificially designed gRNA and Cas protein complex,and design the template tDNA to obtain the desired gene editing by repairing the HDR system in the host cell.This paper aims to use the IB-Svi CRISPR-Cas3 system performs gene editing of target DNA sequences in HEK293T and NIH3T3 cells.,which has been isolated,identified,named,and proven to be capable of self-targeting and non-self-targeting genes in prokaryotic and eukaryotic microorganisms.Human embryonic kidney cells HEK293T and mouse fibroblasts 3T3,which are target cell research,are model mammalian cells,which are characterized by rapid growth,easy transfection,and suitable for intracellular expression.The experiment first explored and mastered the editing operations in mammalian cells using the non-base optimized Type I-B-Svi CRISPR/Cas3 system and the genetic editing tool of the commercial CRISPR/Cas9 system.The drosha gene in 293T cells(target:Exon4)and the vegfa gene in 3T3 cells(target:Exon l)gene editing,g-drosha based on hcas3 and t-drosha::egfp with homologous arm sequences on both sides of the target site and g-vegfa based on scas3 and hcas9,and homologous arms on both sides of the target site were designed.The sequence of t-vegfa::egfp,and cas3/cas9,g-drosha/g-vegfa and t-drosha::egfp/t-vegfa::egfp are ligated into the CRISPR/Cas plasmid.The gene editing plasnid was obtained after transformation amplification verification.The plasmid was introduced into the cells by transient transfection of the liposome,and the editing effect was verified by green fluorescent,specific PCR and sequencing after screening by puromycin,cell morphology and homologous exchange.The results showed that:1)the gene editing plasmid CRISPR-hCas9-t/g-vegfa::egfp In the editing of the vegfa gene of 3T3 cells,the PCR product of the target site cross-primer of the recombinant cell genome and sequencing analysis proved that the target site was obtained correct gene editing;in the gene editing plasmid CRISPR-sCas3-t/g-vegfa::egfp targeted gene editing,the specific cross-primer for PCR did not get the correct band;indicating:base optimization of cas3 gene May be necessary.2)gene editing plasmid AIO-h.Cas3-t/g-drosha::egfp(hca.s3 and mcherry sequence linked by P2A;Cas3 nuclear localization signal unchanged)constructed by hcas3 after cas3 base optimized,The correct results of red,green fluorescence observation,cross-primer PCR,target and off-target sequencing analysis indicated that the gene-editing plasmid AIO-hCas3-t/g-drosha::egfp constructed by base-optimized hcas3 can be used for 293T.Cells perform precise genetic editing.In short,this study successfully achieved the first time that the editing system spanned from the microbial field to the mammalian cell field;it laid a solid foundation for the application of the I-B-Svi type CRISPR-Cas3 system in basic life science and clinical medicine.The editing system will have great application value because of its small molecular weight,non-animal microbial source and no off-target phenomenon.