Abstract
Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disease caused by WAS protein (WASP) mutations. WAS is associated with symptoms including microthrombocytopenia, eczema, autoimmunity and cancer. The molecular mechanism underlying WAS remains elusive. The genotype-phenotype relationship in WAS is complex. There are over 200 mutations that lead to hypomorphic levels or complete loss of WASP, while it is impossible to predict clinical severity based on the mutation alone. To help evaluate phenotype variability due to mutational background of different patients, we developed an isogenic WASP-knockout (WASP-KO) induced pluripotent stem cell (iPSC) model using the CRISPR/Cas9 technique that completely removed the WAS gene. The isogenic iPSC model was differentiated into macrophages, which are reported to be affected by WASP mutations. The results showed complete knockout of WASP did not affect the iPSC pluripotency or efficiency of hematopoietic differentiation from iPSCs. However, it resulted in dramatic morphological changed in the derived macrophages. Importantly, complete knockout of WASP impaired the phagocytosis and migration of macrophages, while promoted their proliferation. Consequently, the cell-cycle inhibitors p16 and p21 were downregulated in WASP-KO macrophages, consistent with their higher proliferation. Additionally, the epigenetic regulator EZH2 and splicing factor SRSF2 are overexpressed in WASP-KO macrophages, which indicates that WASP is involved in epigenetic and mRNA splicing events. In short, we successfully generated a de novo WASP-KO iPSC model, and found that WASP plays important roles in phagocytosis, migration and regulation of cell proliferation. This novel WASP-KO iPSC model can recapitulate WAS disease and can be used in drug screening.