Tandem repeat genes editing using CRISPR-Cas9 system in Medicago truncatula

DWF7 gene encodes Δ⁷-Sterol-C₅-desaturase, and it is involved in brassinosteroid biosynthesis. MtDWF7 gene is located on chromosome 6 and has three tandem repeats in the genome of model legume plant Medicago truncatula. Here, we selected two editing targets for the three copies of MtDWF7 gene, by aligning the homologous segments of the three genes’ exon region. A CRISPR-Cas9 vector harboring two editing targets was constructed, and transformed to wild-type M. truncatula through Agrobacterium rhizogenes mediated transformation to generate transgenic hairy roots. The DNA of transgenic hairy roots was isolated individually. DNA samples from 35 transgenic hairy roots were obtained, and confirmed by PCR. After PCR confirmation, among these 35 transgenic hairy roots’ DNA samples, the editing efficiency of target site 1 for three genes was found to be 44.43%, 5.71%, and 34.29%, respectively. The probability of three genes being edited simultaneously at target site 1 was 5.71%, and no gene editing was observed at target site 2. This study elucidated the simultaneous editing of tandem repeat genes using hairy root transformation, and proved that selecting suitable editing sites can help edit three genes at the same time. This suggests novel way for the functional study of tandem repeat genes in the future.