| In recent years,CRISPR/Cas9 gene editing technology has set off a new technological revolution in the field of life sciences.Using this technology,the plant genome can be edited and modified quickly and efficiently.The modified plants can obtain homozygous plants in contemporary times.At the same time,the exogenous vector sequences can be eliminated by selfing or backcrossing,making the edited plants.There is no essential difference from the spontaneous mutation（natural mutation）or artificial mutation of mutant plants in nature.Therefore,it is expected that this technology will be of great significance to the cultivation of new plant varieties in the future.However,there are few reports on the safety evaluation of the unintended effects of plants cultivated with CRISPR/Cas9 gene editing technology.In this study,gene editing technology was used to edit the thousand-grain weight gene TGW6 in rice,and the positive gene editing materials were further selected to construct a series of（HD、HD-I、HD-A,HD-B,HD-C,HD-D,HD-CH and HD-CB）stable gene editing material.Subsequently,based on the transcriptome and metabolome technology,the above-mentioned rice was evaluated for unintended effects,which provided scientific and comprehensive data support for the evaluation of unintended effects of gene-edited rice,and analyzed the potential health risks of the CRISPR/Cas9 system during the editing process.The main findings are as follows:1.Through DNA extraction,PCR amplification and agarose gel electrophoresis,and then using Sanger sequencing to detect the genotypes of the T3 and T5 generations,it is proved that the edited sites can be stably transmitted to the next generation,and the corresponding genotype materials（HD）are screened.,HD-I,HD-C,HD-D,HD-CH and HD-CB).The thousand-grain weight and grain length of HD family materials were measured,and it was found that the thousand-grain weight of HD-I was similar to that of wild-type HD,and the thousand-grain weight and grain length of the gene-edited T3 and T5 generations were both increased by more than 5%and the grain length by more than 3%compared with wild-type HD.It shows that the modification of the rice TGW6 locus by gene editing has the potential to improve rice yield.2.The relative expression levels of Cas gene and TGW6 gene in HD families were analyzed.PCR results showed that no Cas gene was detected in HD,HD-I,HD-C,HD-D,HD-CH and HD-CB.Q-pcr results showed that the relative expression level of TGW6 gene in wild-type HD was significantly higher than that in other treatments,indicating that the relative expression level of the two genes had genetic stability3.Unintended effects of gene-edited rice using RNA Sequencing（RNA-seq）:In this study,transcriptome sequencing of HD family material was performed to compare and analyze HD-I/HD,HD-T3/HD,HD-T5/HD-T3,HD-T5/HD-C,HD-with-Cas/HD,HD without-Cas/HD,HD-with-Cas/HD-without-Cas differences in transcriptome expression of the seven combinations.By performing GO functional analysis on differentially expressed genes,these Differentially expressed genes are mainly classified into three categories according to their functions:biological process,cellular component and molecular function.The significant enrichment analysis of the GO function and KEGG pathway of the differentially expressed genes showed that gene editing did not bring obvious unexpected effects to the experimental rice,and this unexpected effect was safe and could be passaged stably.4.Unintended effects of gene-edited rice using liquid chromatography-mass spectrometry（LC-MS/MS）technology:In this study,principal component analysis（PCA）was performed on the metabolome of the above rice lines,and metabolite annotation,KEGG annotation The results showed that the metabolic pathways involved in rice metabolites in this study were mainly classified into three categories:Metabolism,Genetic Information Processing,and Environmental Information Processing.The most important biochemical metabolic pathways and signal transduction pathways involved in differential metabolites were identified through KEGG pathway enrichment.The detected differential metabolic pathways and differential metabolite accumulation showed insignificant and safe unexpected effects of gene-edited rice.All the contents and results of this study preliminatively proved the reliability of gene editing technology,provided an important basis for the application of CRISPR/Cas9 technology in plant breeding and biotechnology,and provided an important basis for more studies on the unintended effects of gene editing varieties. |
