RESUMEN
Despite advances in nuclease-based genome editing technologies, correcting human disease-causing genomic inversions remains a challenge. Here, we describe the potential use of a recombinase-based system to correct the 140 kb inversion of the F8 gene frequently found in patients diagnosed with severe Hemophilia A. Employing substrate-linked directed molecular evolution, we develop a coupled heterodimeric recombinase system (RecF8) achieving 30% inversion of the target sequence in human tissue culture cells. Transient RecF8 treatment of endothelial cells, differentiated from patient-derived induced pluripotent stem cells (iPSCs) of a hemophilic donor, results in 12% correction of the inversion and restores Factor VIII mRNA expression. In this work, we present designer-recombinases as an efficient and specific means towards treatment of monogenic diseases caused by large gene inversions.
Asunto(s)
Inversión Cromosómica/genética , Factor VIII/genética , Recombinasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Diferenciación Celular , Células Clonales , Evolución Molecular Dirigida , Células Endoteliales/citología , Células Endoteliales/metabolismo , Exones/genética , Células HEK293 , Células HeLa , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Secuencias Invertidas Repetidas/genética , Recombinación Genética/genética , Especificidad por Sustrato , Secuenciación Completa del GenomaRESUMEN
Large-scale RNAi screens are a powerful approach to identify functions of genes in a cell-type-specific manner. For model organisms, genetically identical (isogenic) cells from different cell types are readily available, making comparative studies meaningful. However, large-scale screens in isogenic human primary cells remain challenging. Here, we show that RNAi screens are possible in genetically identical human stem cells, using induced pluripotent stem cells as intermediates. The screens revealed SMARCA4 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 4) as a stemness regulator, while balancing differentiation distinctively for each cell type. SMARCA4 knockdown in hematopoietic stem and progenitor cells caused impaired self-renewal in vitro and in vivo with skewed myeloid differentiation; whereas, in neural stem cells, it impaired self-renewal while biasing differentiation toward neural lineage, through combinatorial SWI/SNF subunit assembly. Our findings pose a powerful approach for deciphering human stem cell biology and attribute distinct roles to SMARCA4 in stem cell maintenance.
