Reconstruction And Application Of Gene Editing System Directed By SpCas9 And SaCas9 In Soybean

Soybean,as an important grain and oil crop,is the agricultural product exhibiting the most prominent contradiction between supply and demand in China.Besides restricting factors such as natural condition,growing structure and growing technology,breeding technology is the primary factor influencing soybean production.Crossbreeding has disadvantages including long time period,low efficiency and poor predictability,nevertheless,molecular breeding can successfully make up for these drawbacks.Combining crossbreeding with molecular breeding will be the future direction of breeding technology.CRISPR/Cas9 directed gene editing technology is not only a basic research tool,but also one of the most useful molecular breeding tools.At present,there are limiting numbers of Cas9 proteins successfully used in soybean gene editing,which cannot cover the gene editing with entire soybean genome.Moreover,the size of commonly used SpCas9 is too large.Based on CRISPR/Cas9 system,this study focuses on the reconstruction and optimization of both Cas9 nuclease and sgRNA(single guide RNA).By applying soybean hairy root transformation system,we have systematically analyzed gene editing efficiency,mutation characteristic and preference of target gene for SpCas9,SaCas9,split Cas9 protein and SaCas9 with sgRNA-SpCas9 scaffold in soybean.The main results are as follows:1.Soybean gene knockout vectors directed by SpCas9,SaCas9,split-SpCas9 and split-SaCas9 have been successfully constructed.Based on soybean gene editing vectors directed by SpCas9 and SaCas9,we have successfully constructed vectors’ backbones for split-SpCas9,split-SaCas9,SaCas9-SpScaffold and split-SaCas9-SpScaffold through reconstructing and optimizing both Cas9 nuclease and sgRNA scaffold.Then eight sgRNAs targeted soybean genes including mi R156,mi R396 A,SSAC1 and WRKY27,were ligated to backbones of six vectors,leading to 48 gene knockout vectors.2.Positive transgenic soybean hairy roots have been obtained.By applying agrobacterium rhizogenes-mediated transformation of soybean hairy roots,48 gene knockout vectors have been transformed into soybean.The transgenic positive rate was found to be more than 90% by means on PCR tests on DNAs of hairy roots.Finally,15 positive transgenic hairy roots have been achieved for each gene knockout vector.3.PCR amplicons for target gene and their associated next-generation sequencing data have been achieved.The DNA of each positive transgenic hairy root has been applied as template,and barcoding PCR has been executed to amplify target gene.Subsequently,we performed next-generation sequencing on PCR amplicon.Our results showed that the number of valid reads for each sample is equal to 100 at least.Moreover,rates of valid reads were greater than 40%,and sequencing data covered all 720 hairy roots.4.SaCas9,split-SpCas9 and split-SaCas9 exhibit robust editing activities in soybeanBy comparing gene editing efficiencies between different editors,we found that the gene editing efficiencies of SaCas9 and SpCas9 are comparable overall.Nevertheless,SaCas9 presented better performance on mi RNA loci,and SpCas9 presented better performance on protein-coding gene loci.In addition,although both split-SpCas9 and split-Cas9 have lower editing efficiencies comparing to their own full-length Cas9 protein overall,they shown comparable editing efficiencies to their own full-length Cas9 protein on mi RNA loci.5.Both SaCas9-SpScaffold and split-SaCas9-SpScaffold could not work effectively for gene editing in soybeanNot only full-length SaCas9 protein but also split-SaCas9 protein could not use the sgRNA Scaffold of SpCas9 to produce the effective gene editing,which suggests that sgRNA Scaffold of SpCas9 could not be recognized by SaCas9 and split-SaCas9.6.High proportions of insertion were produced by split-SpCas9 and split-SaCas9By analyzing mutation types generated by four editors containing SpCas9,SaCas9,split-SpCas9 and split-SaCas9,we found that the proportion of deletion is higher than that of insertion.However,both split-SpCas9 and split-SaCas9 generated higher proportion of insertion comparing to their own full-length Cas9 protein,especially,the rate of insertion induced by split-SaCas9 was up to 48.45%.The results of this study enrich soybean gene editing tools and further expand the editing scope of CRISPR/Cas9-directed genome editing in soybean.In addition,split-Cas9 proteins present robust editing activities,which lays the foundation for gene editing mediated by viral vector in soybean.