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Tobacco Enzymes Inactivated via Multiplex CRISPR-Cas9 Strategy
Posted by: Prof. Dr. M. Raupp (IP Logged)
Date: April 20, 2017 01:51PM

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: ?
(1,2)-xylosyltransferase (XylT) and ?(1,3)-fucosyltransferase (FucT).

A team of researchers led by S?bastien 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 ?(1,2)-xylose and ?
(1,3)-fucose, while two lines were completely devoid of them, indicating
[] 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.


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