Abstract
Wiskott-Aldrich syndrome (WAS) is a recessive X-linked inmmunodeficiency caused by loss-of-function mutations in the gene encoding the WAS protein (WASp). WASp plays an important role in the polymerization of the actin cytoskeleton in hematopoietic cells through activation of the Arp2/3 complex. In a previous study, we found that actin cytoskeleton proteins, including WASp, were silenced in murine erythroleukemia cells defective in differentiation. Here, we designed a CRISPR/Cas9 strategy to delete a 9.5-kb genomic region encompassing the Was gene in the X chromosome of murine erythroleukemia (MEL) cells. We show that Was-deficient MEL cells have a poor organization of the actin cytoskeleton that can be recovered by restoring Was expression. We found that whereas the total amount of actin protein was similar between wild-type and Was knockout MEL cells, the latter exhibited an altered ratio of monomeric G-actin to polymeric F-actin. We also demonstrate that Was overexpression can mediate the activation of Bruton's tyrosine kinase. Overall, these findings support the role of WASp as a key regulator of F-actin in erythroid cells.