CRISPR/Cas9-Based Mutagenesis of Starch Biosynthetic Genes in Sweet Potato (Ipomoea Batatas) for the Improvement of Starch Quality.

ABSTRACT

CRISPR/Cas9-mediated genome editing is a powerful technology that has been used for the genetic modification of a number of crop species. In order to evaluate the efficacy of CRISPR/Cas9 technology in the root crop, sweet potato (Ipomoea batatas), two starch biosynthetic pathway genes, IbGBSSI (encoding granule-bound starch synthase I), and IbSBEII (encoding starch branching enzyme II), were targeted in the starch-type cultivar Xushu22 and carotenoid-rich cultivar Taizhong6. I. batatas was transformed using a binary vector, in which the Cas9 gene is driven by the Arabidopsis AtUBQ promoter and the guide RNA is controlled by the Arabidopsis AtU6 promoter. A total of 72 Xushu22 and 35 Taizhong6 transgenic lines were generated and analyzed for mutations. The mutation efficiency was 62-92% with multi-allelic mutations in both cultivars. Most of the mutations were nucleotide substitutions that lead to amino acid changes and, less frequently, stop codons. In addition, short nucleotide insertions or deletions were also found in both IbGBSSI and IbSBEII. Furthermore, a 2658 bp deletion was found in one IbSBEII transgenic line. The total starch contents were not significantly changed in IbGBSSI- and IbSBEII-knockout transgenic lines compared to the wild-type control. However, in the allopolyploid sweet potato, the IbGBSSI-knockout reduced, while the IbSBEII-knockout increased, the amylose percentage. Our results demonstrate that CRISPR/Cas9 technology is an effective tool for the improvement of starch qualities in sweet potato and breeding of polyploid root crops.