dCas9-directed DNA demethylation of 35S enhancers to prevent transcriptional gene silencing in transgenic Nicotiana tabacum BY-2 cells

Details

Supervisors

Chaumont, François

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

BY-2 plant cell suspension cultures are a promising platform for glycoprotein production. They offer numerous advantages, but production yields are often impaired by a phenomenon called "RNA silencing". It is a defense mechanism against the expression of foreign DNA. Two interconnected phenomena are involved in RNA silencing. The post transcriptional gene silencing (PTGS) leads to the degradation of mRNAs while the transcriptional gene silencing (TGS) prevents the binding of the RNA polymerase Pol II through the methylation of the promoter. RDR1/6 KO mutants have been previously obtained from silenced cell lines to release transgenes expression from PTGS. Here we demonstrate that TGS still prevent mCherry expression into the RDR1/6 KO cell lines. It can be released using of a chemical DNA demethylating agent. However, this treatment is highly toxic for the cells because of a lack of specificity. In this Master’s thesis, we consider an alternative strategy to induce specific DNA demethylation within 35S enhancers which are commonly used to increase promoter activity. To achieve this, we tested the dCas9-SunTag/TET1cd system. This approach involves of a deactivated Cas9 (dCas9) targeting the 35S enhancer. Besides, the SunTag allows the recruitment of multiples copies of the TET1 catalytic domains (TET1cd) through scFv antibody to induce cytosine demethylation. Cell lines containing the dCas9-SunTag/TET1cd machinery were generated. The expression of dCas9 and TET1cd was verified by Western blot. A cell line exhibiting a robust expression of dCas9-SunTag and scFv-TET1cd was transformed with a construct encoding the reporter gene SecGusPlus under PMA4 promoter with two modified 35S enhancers and a cassette encoding 35S-specific gRNAs. In the future, it would be necessary to quantify the expression level of secGusPlus in those new cell lines to assess the effect of the dCas9-SunTag/TET1cd machinery on transgene expression.