Hi, I am currently working on the silencing of two genes using the method described in '' A Robust CRISPR Interference Gene Repression System in Pseudomonas". This method is based on a binary system with an sgRNA which is encoded by a plasmid under the control of a Ptet and Cas9 is encoded by the chromosome under the control of a Plac or Ptac. The expression of sgRNA is supposed to be constitutive. In my first trials I induced cas9 expression with 1mM IPTG, I have obtained a 2 fold decrease in gene expression. I have increased the IPTG concentration to 50mM concentration considered optimal, I am still at a reduction of 2 fold. Is it necessary to induce the expression of the sgRNA so that the concentration of sgRNA is not a limit for the silencing?
I would like to have feedback from those who have already started this method in pseudomonas aeruginosa.
I wonder have you tried different sgRNAs? I'm not familiar with your model organism but with my model, which is E.coli, the repression efficiency can be very different using with different sgRNA targeting the same gene.
It's possible that the maximum repression efficiency of your sgRNA is two fold. No matter how you tune the expression, it won't drastically change the repression efficiency.
I suggest to try different sgRNA in case you haven't done yet.
- Badges
- Science topic