Construction Of Engineeringed Vero Cells Through Site-directed Knock-in Siat7e Gene By Homogeneous Targeting Mediated By CRISPR/Cas9

Vero cells are the main host cells of many viruses such as New Coronavirus,avian influenza virus,reovirus,adenovirus,canine parvovirus,and cat granulocytic deficiency virus.However,it is difficult to domesticate them into suspension culture cells by traditional methods.Studies have shown that MDCK cells are introduced into human cells ?-2,6-sialyltransferase(st6galnac5)gene(siat7e for short)can reduce the adhesion of medium and strengthen the proliferation of influenza virus into suspension culture cells to produce vaccine.In this study,CRISPR / cas9 mediated gene targeting technology was used to knock human siat7 e gene into Vero cells ?-The promoter region of 2,6-sialyltransferase genome improved the site-specific integration efficiency of foreign genes,constructed and obtained engineering cells expressing target genes,and laid a foundation for its suspension domestication culture.The following results were obtained:1.BGH poly A terminator(369 bp)?EF1-GFP-puro(2300 bp)?Amp R-ori(2184 bp)and T2A(150 bp)sequence were used to get the Gene targeting skeleton vector PBGT2A(4 799 bp)by gene splicing by overlap extension PCR.The genome of green monkey ?-2,6-sialyltransferase gene promoter was amplified by PCR,the homologous long arm(26L)of was about 2 272 bp and the homologous short arm(26S)was about1 068 bp.The 5 064 bp targeting gene fragment 26L-siat7e-Ds Red-26 S was constructed and subcloned into PBG-T2 A.The 9 863 bp gene targeting vector PBG-T2A-26Lsiat7e-Ds Red-26 S was obtained.Sequencing identified that its homology was 100%.2.Five sg RNA target site sequences and five pairs of annealing primer sequences were designed for the gene sequence of the promoter region of the genome of green monkey ?-2,6-sialyltransferase gene.Five sg RNA transcription vectors p GL3-U6-sg RNA-PGK-puromycin-sg RNA1?-sg RNA2?-sg RNA3?-sg RNA4?-sg RNA5 were constructed.Sg RNA transcription vector and Cas9 vector were co transfected into Vero cells,after screening 72 h by puromycin(8 ?g/ ml)and blasticidin(4 ?g/ m L).The Cas9 protein guided by p GL3-U6-sg RNA-PGKPuromycin-sg RNA2?-sg RNA3?-sg RNA5 transcription vector could cleave the target genome sequence.The cleavage efficiency was 37.12%,28.98% and 23.31%,respectively.3.Among the mutations caused by homologous arm cleavage by Ccas9 protein guided by sg RNA,the mutation range of sg RNA-2 is small,mainly before and after the target site.The mutation types include deletion and base insertion mutation;Sg RNA-3mutation region is mainly concentrated near PAM,and different types of mutations exist;The mutation form of sg RNA-5 is single,mainly the deletion of T base.4.Three sg RNA-positive plasmids p GL3-U6-sg RNA-PGK-puromycin,Cas9 vector p ST1374-NLS-flag-linker-Cas9 and gene targeting vector PBG-T2A-26Lsiat7e-26S-Ds Red were co-transfected into Vero cells,8ug/u L Puromycin and 4ug/u Ll blasticidin were screened for 48 hours and three positive engineering cell lines were obtained.The transfection efficiency of sg RNA-2,sg RNA-3,and sg RNA-5 mediated gene targeting vectors were 5% and 8 respectively.%,10%.PCR detection can both amplify the 1011 bp siat7 e gene and 756 bp GFP gene,and successfully obtain genetically engineered cell lines.In this study,a 9 863 bp gene targeting vector including the left and right homology arms,the target gene siat7 e,and the screening gene frame was constructed using SOE PCR and other technologies.At the same time,five sg RNA transcription vectors were designed and constructed based on the gene sequence of the promoter region of the ?-2,6-sialyltransferase gene of the African green monkey genome.T7E1 enzyme digestion test showed that the transcription vectors sg RNA-5,-2,and-3 cut the target genome sequence with efficiencies of 37.12%,28.98%,and 23.31%,respectively.Three plasmids,Cas9 vector and gene targeting vector were co-transfected into Vero cells.Puromycin and blasticidin were screened and verified to obtain three genetically engineered Vero cell lines.