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

• Published in: Turkish journal of biology 2024-01, Vol.48 (5), p.290-298
• Main Authors: Erol, Özgür Doğuş, Şenocak, Şimal, Özçimen, Burcu, Güney Esken, Gülen, Kiliç, Hasan Basri, Kocaefe, Çetin, VAN Til, Niek P, Aerts Kaya, Fatima
• Format: Article
• Language: English
• ISSN: 1300-0152; 1303-6092; 1303-6092; 1300-0152
• DOI: 10.55730/1300-0152.2705

Griscelli Syndrome Type 2 (GS-2) is a rare, inherited immune deficiency caused by a mutation in the 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 gene of GS-2 patient-derived mesenchymal stem cells (MSCs) and induced pluripotent stem cells. We assessed 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 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 (