A Massively Parallel CRISPR-Based Screening Platform for Modifiers of Neuronal Activity
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Abstract
Understanding the complex interplay between gene expression and neuronal activity is crucial for unraveling the molecular mechanisms underlying cognitive function and neurological disorders. In this study, we develop pooled screens for neuronal activity, using CRISPR interference (CRISPRi) and the fluorescent calcium integrator CaMPARI2. Using this screening method, we identified 153 genes associated that changed synaptic excitability in human iPSC-derived neurons, revealing potential links to neurodegenerative and neurodevelopmental disorders. These genes include known regulators of neuronal excitability, such as TARPs and ion channels, as well as genes associated with autism spectrum disorder (ASD) and Alzheimer’s disease (AD) not previously described to affect neuronal excitability. This CRISPRi-based screening platform offers a versatile tool to uncover molecular mechanisms controlling neuronal activity in health and disease.
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This is a powerful and thorough study that uses genetic perturbation to identify human disease genes that control neuronal activity. It's an excellent starting point for untangling the underpinnings of neurological disorders and I'm excited to see how this platform can be used for identifying new therapeutic targets and drug discovery!
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We selected 50 mM KCl treatment as it resulted a CaMPARI2 response with large dynamic range
I love that you used a second mechanism of activation, and optimized it for the best signal in your assay. It seems powerful and gives me confidence in your results!
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lead to sustained activation of Ca2+ channels and lead to an increase in CaMPARI2 signal in our screen
This result is so interesting, it made me rethink what "excitability" means in your screen. I was thinking of CaMPARI2 as the integral of action potentials, but these results suggest to me that there may be several mechanisms which could cause a high CaMPARI signal. For instance, if protein knockdown impaired calcium transport into organelles could this also cause a high CaMPARI signal? If so, how do you interpret the results of the screen, their relationship to neuronal excitability, and to disease?
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he low (blue) and high (red) CaMPARI2 ratio
I was a little surprised here to see HTT listed as a Parkinson's disease gene, I think of it as associated with Huntington's disease! Could you share a little more about it's relationship to Parkinson's, particularly in this case where you have a loss of function?
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neuronal excitability is influenced by diverse molecular pathways
It's awesome you could validate your approach identifying known excitability genes. Did your library include common ion channels like potassium leak channels or voltage-gated sodium channels? It would be a nice additional proof of principle and many of these channels have disease implications.
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neurotransmitter receptor activity, ion transport, and synaptic transmission
This is such a cool screen, and I'm really excited by the hits you found! It seems like there may be a relationship between your top hits (e.g. voltage gated calcium channels) and your calcium sensor as a readout. Do you think this could introduce bias? Is there a way to compensate for this?
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