Restoring the Balance: CRISPRa-Driven β-Tubulin Compensation as a Strategy for Tubulinopathy Treatment

Microtubules are essential cytoskeletal components comprising alpha- and beta-tubulin proteins that facilitate organelle positioning, cell migration, division, and intracellular trafficking. Mutations in tubulin genes can lead to tubulinopathies, a class of rare genetic neurodevelopmental disorders characterized by a range of brain malformations and other clinical features. Recent studies have shown that pathogenic variants in beta-tubulin genes such as TUBB^G308S have been found to underlie the development of ciliopathies, disorders impacting cilia, important organelles for development and cell motility. Thus, mutations in distinct tubulin genes, which present a significant hurdle for the development of therapeutic gene editing strategies and targeted therapeutics. Here, we describe the development of a mutation-independent treatment strategy based on the upregulation of non-mutated beta-tubulin isotype protein using CRISPR-Cas9 activation. By increasing the expression of various beta-tubulin proteins, we demonstrate a restoration of the microtubule network and primary cilia formation.