I am looking to model how expression of a target gene from a state of dysfunction (i.e., knockout; CRISPR-Cas9 induced) to overexpression may influence aspects of neuronal functioning/morphology using patient-derived hiPSCs. The gene itself is associated with several neurodevelopmental phenotypes, so I would like to measure the effects from the iPS cell state -> NPCs -> neuronal state to try and capture whether the degree of expression influences the ability of the cells to mature into neurons.
The target gene itself is quite large (>195kb), so it was suggested to me to apply CRISPRa to achieve overexpression as this makes use of endogenous transcriptional machinery to upregulate the target. However, I have not come across any published articles where (1) gene upregulation is initiated prior to and sustained throughout the differentiation process (i.e., the CRISPRa system is introduced once the cells are at the desired cellular phenotype), or (2) upregulation is maintained over an extended time course. The latter may be necessary to allow me to measure functional outputs of interest. I am thinking an inducible system approach would be useful here, but am open to suggestions!
I am very green in this area of research, and CRISPRa has not been previously attempted in my lab, so would immensely appreciate any advice/recommendations on how I might approach this!
Thanks :)
Dear Kealan Pugsley
I think this publication will give useful ideas for what you are trying to acheive for your project- Article CRISPR Interference Efficiently Induces Specific and Reversi...
Instead of the repressor domain you could plan for activator (eg VP64) domain and use the gRNA for your target.
Best wishes
Shambhabi
Thank you for your suggestion Shambhabi Chatterjee!
It may be the case that I cam unable to effectively upregulate the target gene expression during the differentiation process due to to a reduction doxycycline response.
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