CRISPR knock-out of genes encoding secreted serinecarboxypeptidases in Nicotiana tabacum BY-2 cells

Details

Supervisors

Chaumont, François ; Moens, Mathilde

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité approfondie

Abstract

This master thesis explores the application of the CRISPR-Cas9 gene-editing technology to inactivate 15 serine carboxypeptidase genes (SCPs) in Nicotiana tabacum BY-2 cells. These SCPs, identified through mass spectrometry analysis of the extracellular medium and the cell wall, may be implicated in proteolytic degradation that reduces recombinant glycoprotein yields. BY-2 cells, derived from the tobacco cultivar Bright Yellow-2, are a model system widely used in plant biotechnology due to their rapid growth, scalability, and ability to produce complex recombinant proteins. The inactivation involved designing single-guide RNAs (sgRNAs) targeting 15 SCP genes encoding proteins identified in a proteomic analysis. Homoeologs, resulting from the tetraploid genome of N. tabacum, were carefully analyzed to optimize sgRNA design for both specificity and efficiency. A Golden Gate cloning approach was used to assemble multiplex constructs containing Cas9, sgRNAs, fluorescent marker, and selection resistance genes, creating both single-target and dual-target strategies for gene editing. The constructs were subsequently introduced into BY-2 cells via Agrobacteriummediated transformation. Despite repeated transformation attempts and protocol optimization, including increased kanamycin concentrations for selection, verification of plasmid integrity via restriction digests and sequencing, and testing alternative BY-2 cell lines, no calli were successfully generated. These results led to an investigation into potential barriers to transformation, including plasmid toxicity, incomplete T-DNA transfer or the inactivation of genes involved in development and cellular stress responses. To validate construct functionality, transient expression assays were performed in Nicotiana benthamiana leaves using agroinfiltration. Western blot analyses confirmed the expression of Cas9 and mCherry, indicating that the constructs were functional and capable of expressing the intended proteins in a plant system. This work highlights that achieving stable gene inactivation in plant suspension cells is a complex process. It also points out the need to better identify non-essential SCP genes to avoid lethal inactivation.