Construction of tomato plants with suppressed endo-?-N-acetylglucosaminidase activity using CRISPR-Cas9 mediated genome editing

High mannose-type free N-glycans with a single N-acetyl-D-glucosamine (GlcNAc) residue at the reducing end (GN1-HMT-FNGs) are produced by cytosolic endo-p-N-acetylglucosaminidase (EC:3.2.1.96) (ENGase) and are ubiquitous in differentiating and growing plant cells. To elucidate the physiological functions of HMT-FNGs in plants, we identified the ENGase gene in tomato (Solyc06g050930) and detected ENGase activity and increased production of GN1-HMT-FNGs during tomato fruit maturation. However, the precise role of GN1-HMT-FNGs in fruit maturation remains unclear. In this study, we established tomato ENGase mutants with suppressed ENGase activity via CRISPR/Cas9 genome editing technology. DNA sequencing of the Aeng mutants (T0 and T1 gener-ations) revealed that they had the same mutations in the genomic DNA around the target sequences. Three null CRISPR/Cas9 segregant plants of the T1 generation (Aeng1-2,-22, and-26) were used to measure ENGase activity and analyze the structural features of HMT-FNGs in the leaves. The Aeng mutants did not exhibit ENGase activity and produced GN2-HMT-FNGs bearing tow GlcNAc residues at the reducing end side instead of GN1-HMT-FNGs. The Aeng mutants lack the N-terminal region of ENGase, indicating that the N-terminal region is important for full ENGase activity. The fruits of Aeng mutants (T2 generation) also showed loss of ENGase ac-tivity and similar structural features of HMT-FNGs of the T1 generation. However, there was no significant difference in fruit maturation between the T2 generation of the Aeng mutants and the wild type. The Aeng mutants rich in GN2-HMT-FNGs could be offered as a new tomato that is different from wild type containing GN1-HMT-FNGs.

Automated summary

Through CRISPR/Cas9 technology, researchers have established tomato mutants with suppressed ENGase activity. These mutants lack ENGase activity, resulting in the production of GN2-HMT-FNGs with a different structure instead of the ubiquitous GN1-HMT-FNGs. However, there is no significant difference between the mutants and the wild type during fruit maturation.