Study On Virus - Mediated CRISPR / Cas9 Tobacco Gene Editing System

Genome editing is a technology producing DNA double strand breaks at a specific position of the genome usingthe nucleic acid enzyme with specific nucleic acid sequences, leading to activation of cell repair mechanism non homologous end joining or homologous recombination, gene knockout, chromosome recombination and gene targeted insertion or substitution. When there is exogenous DNA or virus, it will activate the system, and the CRISPR/Cas system will break the exogenous DNA or virus. In order to get the immune system, the CRISPR/Cas system will be integrated by the fragments ofinvasion sequence, and then carry out the degradation of homologous sequences to the specific crRNA. This system is simple, high precision, low cost, and can be used to edit multiple sites at the single experiment.This study was to explore the expression of crRNA and Cas9 protein, the key factors of CRISPR/Cas9 system, in tobacco with the early Brown virus, and to evaluate the effect of tobacco gene editing based on the method. The main results are as follows:1, The target sequence of PDS gene was design based on the homologous sequences of PDS gene in NCBI tobacco for obtaining crRNA. The construct pCAPE2: crRNA joined pCAPE2-GFP and crRNA. Firstly, the transgenic lines expressing Cas9 protein were produced using the Agrobacterium transformation method. Secondly, the plasmids pCAPE1 and pCAPE2: crRNA were transformed into Agrobacterium GV3101, and the Agrobacterium pCAPE1 and pCAPE2: Cas9-crRNA infected the transgenic lines with the mixture 1: 1, but the genome editing phenotype of photo bleaching can’t be observed in the lines infected.2, The sepcial target sequence was designed based on the nucleotide acid sequence of the PDS gene of tobacco, and their complementary nucleotide acids was synthesized with restriction endonuclease sites added. The primes with restriction endonuclease sites was designed for amplifying the coding region of Cas9. The annealing products, Amplification product and vector were digested with endonucleases, and then three segments were joined together with T4 ligase. The vector pCAPE2: Cas9-crRNA was conformed with sequencing technology. The plasmids pCAPE1 and pCAPE2: Cas9-crRNA were transformed to Agrobacterium GV3101 respectively. After cultivation, the Agrobacterium containing pCAPE1 and pCAPE2: Cas9-crRNA were used to the infection of tobacco variety Samsun with proportion 1:1. The Photo bleaching phenotype was discovered in some regions ofnew leaf of tobacco at 2-3 weeks after injection, which indicated that pCAPE1 and pCAPE2 carrying Cas9 and crRNA fragments can infect tobacco, and reproduce in the whole plant. The genomic DNA was extracted for the region of photo bleaching,and the specific primers of amplifying the segment containing target sequences was used for PCR reaction with genomic DNA as template. The amplification products were connected to pGEM-Teasy. The sanger sequencing provided that the PDS gene of tobacco variety Samsun was found to be different from the Target sequence of the designed CRISPR/cas9 system.3, The promoter of gamete-specific expression gene NtDMC1 was isolated and was inserted into the expression vector, and then Cas9 was inserted into the down stream of the promoter using Gateway technology. pH7WG: Nt DMC1: Cas9 was obtain for expressing the Cas9 gene specially in gamete.This paper produced the constructs pCAPE2:crRNA and pCAPE2: Cas9-crRNA mediating the expression of the key factors Cas9 and crRNA by viral PEBV. The transgenic lines expressing Cas9 protein were obtained. The promoter of gamete-specific expression gene NtDMC1 was isolated and pH7WG: NtDMC1: Cas9 was constructed for expressing the Cas9 gene in gamete specially. The genome editing experiments showed that PEBV can induce the expression of Cas9 and crRNA, which should provide some theoretical foundation for the genomic editing of CRISPR/cas9 system mediated by virus.