Tobacco Enzymes Inactivated via Multiplex CRISPR-Cas9 Strategy

Plants or plant cells can be used to produce pharmacological glycoproteins such as antibodies or vaccines. However, these proteins carry N-glycans with plant-typical residues, which greatly impact the effect or activity of the protein. Two enzymes are responsible for the addition of plant-specific glycans: β(1,2)-xylosyltransferase (XylT) and α(1,3)-fucosyltransferase (FucT).

A team of researchers led by Sébastien Mercx from Catholic University of Louvain aimed to knock-out two XylT genes and four FucT genes in Nicotiana tabacum BY-2 suspension cells using CRISPR-Cas9. Three XylT and six FucT sgRNAs were designed to target conserved regions. After transformation of N. tabacum BY-2 cells, genome-edited lines were obtained and their protein complements were analyzed.

Three lines showed a significant reduction in β(1,2)-xylose and α(1,3)-fucose, while two lines were completely devoid of them, indicating complete gene inactivation. The KO lines did not show any particular morphological changes and grew similarly as the wild types.

One KO line was then transformed with genes encoding a human IgG2 antibody. The IgG2 expression level was as high as in a control transformant which had not been edited.

For more information, read the article in Frontiers in Plant Science.