CRISPR/Cas9-mediated Genome Editing In Soybean

Soybean(Glycine max(L.) Merr.) is an important legume crop with great economic value,it is also the main source of plant protein for human beings. With the growth of the world’s population, the demand of soybean is also increasing. Developing new technologies for soybean genetic improvement is of great significance, especially for researching on gene function and new varieties. Recently,CRISPR/Cas9 system has been successfully used for genome engineering in many important crops.However, this method has not been widely used in soybean. In the present study, we successfully applied the CRISPR/Cas9 system to generate and estimate genome editing on foreign genes and endogenous genes in soybean. Subsequently, we explored the CRISPR/Cas9-mediated genome knockin and replacement. The main results were listed as followed.1 Using the protein sequences of FEI1, FEI2 and SHR of Arabidopsis thaliana as a seed sequence to blast soybean genome database in NCBI, we have found three high homology gene fragments, named Gm FEI1, Gm FEI2 and Gm SHR respectively. We successfully cloned these three genes, as target genes for gene editing.2 In the present study, we described a rapid and highly specific method for generating and detecting CRISPR/Cas9 mediated genome editing in desired target genes in soybean. To improve transformation efficiency, the sgRNA and Cas9 cassettes were assembled on one vector. The Cas9 was codon optimized for dicotyledon plants. Once a desired target sequence was selected, only the DNA sequence encoding sgRNA needs to replace. This vector contained a GFP fluorescent label and greatly improved the efficiency of screening for positive hair roots.3 In the present study, we designed a sgRNA that targeted a transgene(bar) and six sgRNAs that targeted different sites of two endogenous soybean genes(GmFEI2 and Gm SHR). The targeted DNA mutations were detected in soybean hairy roots. The efficiency of each target site was estimated.Targeted DNA mutations were detected in approximately 54% of the 170 transgenic hairy roots, with indel frequencies ranging from 0.6% to 95.0%. PCR/RE assay showed that mutation rates of bar-SP1,Gm FEI2-SP1, GmFEI2-SP2 and GmSHR-SP3 were 36.7%, 60.0%, 93.3% and 35.7% respectively, indel frequencies were 1.3% ~ 21.0%, 0.6% ~ 18.8%, 1.0% ~ 95.0% and 2.8% ~ 28.7% respectively. T7 EI assay showed that mutation rates of GmSHR-SP1 and GmSHR-SP2 were 50.0% and 45.4% respectively,indel frequencies were 2.3% ~ 21.3% and 8.7% ~ 30.0% respectively. Sequencing was subsequently used to detect the type of indels in the targeted sites. Small insertions, deletions and mismatches were detected in these target sites. These results demonstrated that this customized CRISPR/Cas9 system shared the same efficiency for both endogenous and exogenous genes in soybean.4 We also performed experiments to detect the potential of CRISPR/Cas9 system to simultaneously edit two endogenous soybean genes using only one customized sgRNA. We designed a sgRNA, namely Gm FEI2-SP3. Both GmFEI1 and GmFEI2 have this target sequence at the third exon. PCR/RE assayshowed that 3 out of 30 soybean hairy roots which have the expression of sgRNA: Cas9 showed indels at the target sites of both GmFEI1 and GmFEI2 genes at one time, although they had different efficiencies. The indel frequencies in GmFEI1 of these three roots were 32.7%, 20.3% and 21.6%respectively, while the indel frequencies in GmFEI2 of these three roots were 7.0%, 5.4% and 27.0%respectively. Undigested bands were subsequently cloned and sequenced to detect the type of indels in the two genes. Some small insertions and deletions were detected in the target sites. The results demonstrated that CRISPR/Cas9 system could simultaneously edit two endogenous soybean genes using one customized sgRNA.