Research On Editing-site-specific Detection Technology For Genome-edited Plants

Gene editing technology can be used for targeted editing of organisms' genomes,and is widely used in crop breeding due to its high efficiency,precision and low cost.According to the definition of genetically modified organisms(GMOs),genome-edited products meet the characteristics of "altering genome composition by genetic engineering technology" and fall under the scope of the Regulations on the Safety Management of Agricultural GMOs in China.Compared with traditional GM products,genome-edited crops lack exogenous promoters,terminators,and functional genes,so the currently applied transgenic detection strategies,such as universal element screens,gene-specific assays,vector-specific assays,and event-specific assays are not applicable to genome-edited crops.And the existing gene editing assays can only determine whether the target gene is edited,or can only assess the editing efficiency of the target gene,and cannot identify and quantify the specific genome-edited products,which cannot meet the needs of safety regulation of transgenic products.Therefore,in this study,we used seven CAO1-edited and five SP1-edited materials to investigate editing-site-specific detection technology,and established an accurate and efficient quantitative detection method.The main research contents are as follows.1.Sequence identification of target loci for gene editing rice.Sequencing primers were designed on both sides of the rice gene editing locus with amplification lengths of 755 bp(CAO1 gene)and 702 bp(SP1 gene),and the target loci of the rice CAO1-edited and SP1-edited materials were analyzed by PCR amplification,agarose gel electrophoresis,and sequencing of the target strips by cutting and sequencing.Seven homozygous CAO1-edited materials and five homozygous SP1-edited materials were successfully identified.2.Principles of primer probe design.Universal PCR primers were designed on both sides of the editing sites of CAO1 and SP1 genes,and the amplification products were 100?250 bp in length.Editing-site-specific Taq Man probes were designed at the editing sites,and the edit site-specific Taq Man probes were combined with the universal primers respectively to establish the editing-site-specific PCR method.Using CAO1-edited and SP1-edited rice and wild type rice as materials,the specificity and sensitivity of the editing-site-specific PCR methods were tested.Each method was able to specifically identify the corresponding gene-edited material with a detection sensitivity of 5-10 haploid genomes.3.Real-time PCR quantitative assay.The DNA of gene-edited material were mixed with wild-type rice DNA to prepare blind samples with 5%,2%,1% and 0.1% gene editing DNA content.Different primer/probe concentrations were set for real-time PCR amplification of the blind samples to determine the optimal primer/probe concentration.The results of real-time quantitative PCR showed that for the blind samples with 5% gene-edited DNA content,the relative standard deviations(RSD)and Bias of the quantitative results were less than 25%,except for the single-base insertion gene-edited material CAO1-7,which met the performance requirements of GMO analytical method;for the blind samples with 2% gene-edited DNA content,only the quantitative results of the gene-edited material CAO1-2 and SP1-3 met the requirements;The blind samples with 1% and 0.1% gene-edited DNA content could not be accurately quantified by real-time quantitative PCR reaction.4.Digital PCR quantitative assay.The optimal annealing temperature of digital PCR reaction was determined at 55.5°C based on the difference in signal intensity between positive and negative microdroplets.Both single and duplex droplet digital PCR(dd PCR)were used to quantify the blind samples,respectively.The RSD values of the quantification results was less than 25% and the relative bias was in the range of ±25% for all samples except for the blind sample of 0.1% gene-edited material CAO1-2.Two quantification strategies were used for the dupex dd PCR,one is SP1/PLD duplex dd PCR,in which a combination of the editing-site-specific primer/probe set and the reference gene PLD primer/probe set were added to one reaction well to quantify the copy number of both the editing DNA and PLD gene;the other is CAO1(SP1)/W,in which a universal primer,the editing-site-specific probe and the wild type probe were added to the same one reaction well to simultaneously measure the copy number of edited type DNA and wild type DNA.The RSD values of all samples were less than 25% and the relative bias were between ±25%,which met the performance requirements of GMO detection methods.