Abstract
Precise genome editing in combination with viral delivery systems provides a valuable tool for neuroscience research. Traditionally, the role of genes in neuronal circuits has been addressed by overexpression or knock-out/knock-down systems. However, those techniques do not manipulate the endogenous loci and therefore have limitations. Those constraints include that many genes exhibit extensive alternative splicing, which can be regulated by neuronal activity. This complexity cannot be easily reproduced by overexpression of one protein variant. The CRISPR activation and interference/inhibition systems (CRISPRa/i) directed to promoter sequences can modulate the expression of selected target genes in a highly specific manner. This strategy could be particularly useful for the overexpression of large proteins and for alternatively spliced genes, e.g., for studying large ion channels known to be affected in ion channelopathies in a variety of neurological diseases. Here, we demonstrate the feasibility of a newly developed CRISPRa/i toolbox to manipulate the promoter activity of the Cacna1h gene. Impaired, function of the low-voltage-activated T-Type calcium channel CaV3.2 is involved in genetic/mutational as well as acquired/transcriptional channelopathies that emerge with epileptic seizures. We show CRISPR-induced activation and inhibition of the Cacna1h locus in NS20Y cells and primary cortical neurons, as well as activation in mouse organotypic slice cultures. In future applications, the system offers the intriguing perspective to study functional effects of gain-of-function or loss-of-function variations in the Cacna1h gene in more detail. A better understanding of CaV3.2 channelopathies might result in a major advancement in the pharmacotherapy of CaV3.2 channelopathy diseases.
Highlights
MATERIALS AND METHODSThe human genome encodes approximately 400 ion channel genes, encompassing both voltage-gated and ligand-gated ion channels (Hutchings et al, 2019)
The functional characterization of individual ion channels in terms of gain- and loss-of-function approaches has been addressed by the exogenous overexpression of genetically encoded proteins and by knock-down systems via RNA interference (RNAi)/genetic gene ablation, respectively
We present a modular method using a CRISPR activation (CRISPRa)/i system that allows to manipulate the endogenous expression of Cacna1h in vitro and in vivo
Summary
MATERIALS AND METHODSThe human genome encodes approximately 400 ion channel genes, encompassing both voltage-gated and ligand-gated ion channels (Hutchings et al, 2019). Ion channels are pore forming membrane proteins that allow ionic flows across membranes and are crucial for normal functioning of many tissues, including the central and peripheral nervous system, heart, kidney, and liver (Cannon, 2007). Coined as ion “channelopathies,” have been associated with a large variety of diseases. Besides disorders of the nervous system (e.g., epilepsy, ataxia, Alzheimer’s disease, and Autism spectrum disorders), cardiac arrhythmia and several muscle, endocrine, and renal disorders are linked to dysfunction of ion channels (Wang et al, 1996; Heeringa et al, 2009; Ryan et al, 2010). Major obstacles to manipulate ion channels are given by their large size, which limits options for widespread and in vivo overexpression and their diversification by alternative splicing
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
