Correction of Griscelli Syndrome Type 2 causing mutations in the RAB27A gene with CRISPR/Cas9

Griscelli Syndrome Type 2 (GS-2) is a rare, inherited immune deficiency caused by a mutation in the RAB27A gene. The current treatment consists of hematopoietic stem cell transplantation, but a lack of suitable donors warrants the development of alternative treatment strategies, including gene therapy. The development of mutation-specific clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 gene editing technology has opened the way for custom-designed gene correction of patient-derived stem cells. In this study, we aimed to custom design CRISPR/Cas9 constructs and test their efficiency on homology-directed repair (HDR) on the correction of exon 3 and exon 7 mutations in the RAB27A gene of GS-2 patient-derived mesenchymal stem cells (MSCs) and induced pluripotent stem cells. Materials and

The use of CRISPR/Cas9 to genetically correct MSCs and iPSCs from GS-2 patients with different mutations through HDR is feasible but requires optimization of the procedure to reduce cell death and improve stem cell function before clinical application.

We assessed RAB27A gene and protein expression using qRT-PCR, Western Blot, and immune fluorescence in GS-2 patient-derived MSCs and induced pluripotent stem cells (iPSCs). Guide RNAs (gRNAs) and donor DNAs were designed based on patient mutations in exon 3 and exon 7 using the CHOPCHOP online tool and transfected into GS-2 MSCs and iPSCs by electroporation. The cells were cultured for 2 days and then used for mutation analysis using DNA sequencing.

MSCs and iPSCs from the GS-2 patients lacked RAB27A gene and protein expression. After gRNA and donor DNAs were designed and optimized, we found HDR efficiency with gRNA3.3 (10% efficiency) and gRNA7.3 (27% efficiency) for MSCs but lower efficiency in iPSCs (<5%). However, transfection of both MSCs and iPSCs resulted in massive cell death, loss of colony formation, and spontaneous differentiation.