The Scope Of Soybean Genome Editing Was Broadened By Using The SpCas9 Variants

Soybean originates from China and has been widely cultivated around the world,which supplies plenty of protein and oil for human.With the world population growth and the continuously natural environmental deterioration,gene function studies and new varieties breeding in soybean have gotten more and more attentions from researchers.Gene function studies in plant and animal has been largely promoted by the development and application of CRISPR/Cas9 gene editing technology.The traditional SpCas9 can recognize and function on NGG or NAG protospacer adjacent motif(PAM),but distribution of those sequences on genome is limited.The variants of SpCas9 including xCas9 and SpCas9-NG have been found to recognize NG PAM in plant species such as rice and Arabidopsis,but their applications in soybean have not been reported.In order to expand the application of CRISPR in soybean,xCas9,SpCas9-NG and xNGCas9 variant that is produced by joining mutation sites from xCas9 and SpCas9-NG together have been used in this study.sgRNA targeted sixteen PAMs including NG,CAG,GAA and GAT were designed,and CRISPR knockout vectors were constructed based on SpCas9 and three SpCas9 variants.Moreover,Agrobacterium rhizogenes K599-mediated transgene method was applied for getting hairy roots,and then next-generation sequencing technology was used to analyze the gene editing efficiencies for SpCas9 and its variants in soybean hairy roots.Our main results are as followed:1.Construction of soybean gene knockout vectorsUsing the method of overlap PCR,SpCas9 was mutated into xCas9,SpCas9-NG and xNGCas9.A total of 16 PAM types have been designed,including:CGG,CAG,GAT,GAA,AGA,TGA,CGA,GGA,AGC,TGC,CGC,GGC,AGT,TGT,CGT and GGT.Three different target genes were selected for each type to design targets,and a total of 192 knockout vectors were obtained.2.Obtaining soybean genetically modified hairy rootsThe soybean hairy roots transformation method mediated by Agrobacterium was used to successfully transform the above-mentioned vectors into soybeans.Each vector obtained 15 hairy roots,and a total of about 2880 soybean hairy roots were obtained.3.The variants of SpCas9 broaden the editing scope of SpCas9Using NGS technology,gene editing detection of soybean hairy roots DNA,found that SpCas9 variants widened the editing range of SpCas9.The SpCas9 variant extends the PAM sequence of SpCas9 from NGG to NG.In addition,SpCas9-NG can play a role in GAA and GAT,and xNGCas9 can play a role in GAA.Compared with SpCas9,each SpCas9 variant can recognize more kinds of PAM sites,which broadens the range of soybean genome editing mediated by SpCas9.4.Each SpCas9 variant has different editing characteristicsAll variants of SpCas9 can play a role in the genome of soybean hairy root cells,but they have their own characteristics compared to SpCas9.xCas9 can play a role at the NG PAM site,but the efficiency is not high;The NG PAM site has a high editing efficiency;xNGCas9 can also play a role in the NG PAM site,which is more efficient at the NGC site.In summary,this research has developed a new gene editing system suitable for soybean,which not only broadens the editing range of CRISPR/Cas9 in soybean,but also provides powerful molecular tools for soybean gene function research and genetic improvement.