Construction Of RNA Editing Technology Based On CRISPR-Cas13 System To Inhibit TMV

With the advent of the CRISPR-Cas system,gene editing technology has developed rapidly.Prior to the launch of this research,the research on the CRISPR-Cas9 system to directly target and edit DNA has matured,and the development direction of the new field of the CRISPR-Cas system has turned more towards RNA editing.Recent studies have found that the CRISPR-Cas II type VI CRISPR-Cas13 a system can act in plants for resistance to plant RNA viruses.This study clarified the mechanism of the CRISPR-Cas13 a gene editing system for targeted editing of RNA,and constructed a new CRISPR-CAS13 a expression vector that can be used for multi-target editing of the TMV genome.Based on CRISPR-Cas13 a engineering,a natural TMV-resistant transgenic tobacco plant was created,which is a plant The prevention and treatment of RNA viruses provides scientific and reliable scientific theoretical basis.Below are key research findings:1.Construction of a new CRISPR-Lsh-Cas13 a expression vector based on the CRISPR-Cas13 a system for multi-target directed editing of TMV: through sequence analysis of the genomes of 38 different isolates of TMV,the conservative regions of the Rd Rp,MP,and CP genes were selected to meet the requirements Target,design three cr RNAs and connect them in tandem;clone the tandem cr RNAs into the Lsh-Cas13 a expression vector to construct a new CRISPR-Lsh-Cas13 a expression vector that can be used for multi-target editing of the TMV genome.The recombinant vector uses the At U6 promoter to express cr RNA and the UBQ10 promoter to efficiently express the Cas13 a protein,and the upstream contains the kanamycin resistance gene.The connection of the target sequence and the integrity of the recombinant vector are verified by the method of target sequence sequencing and Agrobacterium liquid PCR.The results show that the target sequence has been successfully connected to the Cas13 a expression vector,and the recombinant vector has good integrity.2.Recombinant vector function verification and CRISPR-Lsh-Cas13 a system editing RNA performance verification: express the vector in the plant by the method of Agrobacterium transient infection,carry out the inoculation treatment,and pass the virus biological determination,gene relative quantification and protein level analysis The results showed:(1)The PDS m RNA of Nicotiana benthamiana was selected as the target gene for editing in the positive control experiment of this study,and the results showed that the leaves appeared albino phenomenon.(2)The results of virus biology test showed that the ratio of fluorescent signal of the infected leaves of Nicotiana benthamiana treatment group and the negative control group was about 1:10.44,and the ratio of the number of plaques on the leaves of the dead spot treatment group and the negative control group.About 1:11.(3)The relative quantitative results of q RT-PCR virus gene showed that the ratio of the relative content of TMV-30 b CP gene in the infected leaves of Nicotiana benthamiana treatment group and the negative control group was about 1:7.44,and the difference was extremely significant.According to the relative content ratio of CP gene,it can be seen The inhibitory efficiency of CRISPR-Lsh-Cas13 a system against TMV-30 b was86.56%;the relative content ratio of wild-type TMV Rd Rp gene in the Samsun NN treatment group and the negative control group was about 1:20.16,and the relative content ratio of MP gene was about1:20.16 The relative content ratio of CP gene is 1:10.3,and the relative content ratio of CP gene is about1:7.34.According to the relative content ratio of CP gene,the inhibition efficiency of CRISPR-Lsh-Cas13 a system against wild-type TMV is 86.38%.(4)Western Blot virus protein content detection results showed that TMV was detected in the leaves of the treatment group and the leaves of the negative control group,and the TMV protein content of the leaves of the treatment group was significantly lower than that of the leaves of the negative control group.The results of the positive control test showed that the vector function was normal and could be used in this study;the results of virus biological assays can preliminarily determine that the CRISPR-Lsh-Cas13 a gene editing system can effectively inhibit virus replication and spread,and the relative quantitative results of q RT-PCR virus genes confirmed the initial judgment.As a result,the Western Blot virus protein content test result makes the judgment result more convincing.3.CRISPR-Cas13a-based natural anti-TMV transgenic tobacco plants inhibiting,preventing and controlling TMV effectiveness verification: the transgenic Nicotiana benthamiana and wild-type tobacco test plants were inoculated,and the virus biology test and TMV-30 b CP gene were carried out.The relative quantitative analysis results are as follows:(1)Virus biological assay results show that the ratio of fluorescence signal of infected leaves of the treatment group(transgenic plants)and the negative control group(wild-type Nicotiana benthamiana)is about 1:15.02;(2)The relative quantitative results of q RT-PCR virus gene showed that the ratio of the relative content of TMV-30 b CP gene in infected leaves of the treatment group and the negative control group was about 1:15.21.Based on this data,the inhibitory efficiency of the transgenic plants against TMV-30 b was 93.43%.