Research Of Rice Aroma Gene Editing Using CRISPR/Cas9 Technology And The Methods Detecting Targeted Mutation

Fragrant rice is favored all over the world because of its agreeable scent.Conventional breeding strategies to improve rice productivity and rice quality had difficulty owing to long cycle and heavy workload,and it cannot meet the present production needs.The CRISPR/Cas system has emerged as a robust technology for targeted gene editing in rice and other crops,which is a useful approach to creating important agronomic traits.The presence of a defective BADH2 allele encoding betainealdehydedehydrogenase?BADH2?resultsinthesynthesisof2?acetyl?1?pyrroline?2AP?,which is a major fragrance compound.Here,CRISPR/Cas9 targets editing techniques were engineered to target and disrupt the BADH2 gene.Two 20-nucleotide?nt?regions?Badh2KO1 and Badh2KO2?at the5'end of a protospacer adjacent motif?PAM?in the second exon and the seventh exon of were selected as the editing targets respectively.After integrating the single-guide RNA?sgRNA?and Cas9 cassette in a single binary vector,94 transgenic rice plants harboring sgRNA:Cas9 were generated by A.tumefaciens-mediated stable transformation.By analyzing the targeting site on the genome of corresponding transgenic plants,the mutations were determined.The mutagenesis efficiency of Badh2KO1 target was 27%,while the mutagenesis efficiency of Badh2KO1 target was 63%and 5 lines have no bases change in targets.Stable and homozygous mutants were inherited from T₀ mutant plants for two generations.The mutated lines had various indel mutations,resulting in the change of amino acid sequences in the encoded protein.The 2-AP content of these mutants measured by GC-MS technology was significantly higher than that of wild-type plants.These results indicate that CRISPR/Cas9 can orientate rice genes and alter rice traits.Mutation detection is a necessary step in the study of plant CRISPR/Cas9targeting mutation.In order to compare the characteristics of different detection methods,this study used a rice T₀ generation CRISPR/Cas9 target group as the material,compared the current commonly used restriction endonuclease fragment length polymorphism analysis,T7 endonuclease analysis,single strand conformation Polymorphism analysis,high resolution dissolution curve analysis and direct sequencing analysis of the five means of detection accuracy and sensitivity.Our results show that the positive results of these methods are high,but there are some differences in the detection ability,and then we also discuss the differences between the experimental cycle and the economic cost of different methods.Our work provides a reference for the selection of plant CRISPR/Cas9 target editing methods.