Abstract
Human induced pluripotent stem cells (hiPSCs) represent a powerful tool to investigate neuropathological disorders in which the cells of interest are inaccessible, such as in the Charcot-Marie-Tooth disease (CMT), the most common inherited peripheral neuropathy. Developing appropriate cellular models becomes crucial in order to both study the disease's pathophysiology and test new therapeutic approaches. The generation of hiPS cellular models for disorders caused by a single nucleotide variation has been significantly improved following the development of CRISPR-based editing tools. In this study, we efficiently and quickly generated, by CRISPR editing, the two first hiPSCs cellular models carrying alterations involved in CMT4C, also called AR-CMTde-SH3TC2. This subtype of CMT is associated with alterations in the SH3TC2 gene and represents the most prevalent form of autosomal recessive demyelinating CMT. We aimed to develop models for two different SH3TC2 nonsense variants, c.211C>T, p.Gln71* and the most common AR-CMTde-SH3TC2 alteration, c.2860C>T, p.Arg954*. First, in order to determine the best CRISPR strategy to adopt on hiPSCs, we first tested a variety of sgRNAs combined with a selection of recent base editors using the conveniently cultivable and transfectable HEK-293T cell line. The chosen CRISPR base-editing strategy was then applied to hiPSCs derived from healthy individuals to generate isogenic CMT disease models with up to 93% editing efficiency. For point mutation generation, we first recommend to test your strategies on alternative cell line such as HEK-293T before hiPSCs to evaluate a variety of sgRNA-BE combinations, thus boosting the chance of achieving edited cellular clones with the hard-to-culture and to transfect hiPSCs.