Bsasic Research On Gene Editing Of Nicotiana Tobacco Using CRISPR/Cas9 System

CRISPR/Cas9 is an adaptive immune defense mechanism found in bacteria and archaea.Uptake of exogenous DNA of short fragments lets the host to produce spacer sequences,which mediate acquired resistance against the virus and plasmid.CRISPR/Cas9 system is widely used in gene editing for variety of organisms because of its simplicity,reliability,efficiency and accuracy.In this study,we used Nicotiana Benthamiana as target organism to edit endogenous PDS(Phytoenedesaturase)gene and exogenous GFP(Green Fluorescent Protein)gene using CRISPR/Cas9 system.Both transient expression and genetic transformation system for CRISPR/Cas9 mediated editing were established,laying a foundation for the study of biological function of other endogenous genes in plant in the future.Three target sequences were predicted for the endogenous PDS gene and four target sequences were predicted for the exogenous GFP gene.The corresponding guide sequences for these target sites were designed,and the binary overexpression vectors were constructed.And the intermediate cloning vector psgR-Cas9-At was successfully transformed into a In-Fusion cloning vector psgR-Cas9-IF-At which is highly operational.The constructed recombinant binary expression vector was transformed into Agrobacterium tumefaciens and injected into the tobacco leaves by injection.The recombinations offour guide sequences for FP gene were transformed into Agrobacterium tumefaciens and injected leaves of the transgenic Nicotiana benthamiana 16C-NB(16C transgenic Nicotiana benthamiana,16C-NB),which overpress GFP gene,the editing effects of CRISPR/Cas9 is evaluated by PCR-RE method.The result shows that only GFP-guide-1 and GFP-guide-3 have editing effects.The recombinations of three guide sequences for PDS gene were transformed into Agrobacterium tumefaciens and injected leaves of the wild type Nicotiana benthamiana 17WT-NB(17 Wild Type Nicotiana benthamiana,17WT-NB),the editing effects of CRISPR/Cas9 is evaluated by RE-PCR method.The result shows that only PDS-guide-1 has editing effects.In addition to single site editing,multiple site editing with PTG techniques that 3 guide sequences of PDS gene assembled with two guide sequences respectively(PTG-1+2,PTG-1+3,PTG-2+3)was also tested.The assembled guide sequences were constructed into binary expression vector and the constructed recombinant binary expression vector was transformed into Agrobacterium tumefaciens and injected into the leaves of 17WT-NB by injection.The transient expression of PTG cassette in tobacco leaf editing effects evaluated by PCR method,and the result shows that only PTG-1+2 has editing effects.Based on above works,Agrobacterium tumefaciens-mediated leaf disc transformation experiments were carried out for PDS-guide-1,in order to obtain transgenic plants of single site editing.Agrobacterium mediated transformation of PDS-guide-1 produced 99 transgenic lines,among which 45 showed positive in PCR detection,but survived positive lines are 22,which laid the foundation for the editing of plants with stable inherited PDS gene.