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Turkish Journal of Biology

Abstract

Background/aimGriscelli Syndrome Type 2 (GS-2) is a rare, inherited immune deficiency caused by a mutation in the RAB27A gene. Current treatment consists of hematopoietic stem cell transplantation. Lack of suitable donors warrants the development of alternative treatment strategies, including gene therapy. The development of mutation-specific CRISPR/Cas9 gene editing technology has opened the way for custom designed gene correction of patient-derived stem cells. Here, we aimed to custom design CRISPR/Cas9 constructs and test their efficiency of 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 methodsWe assessed RAB27A gene and protein expression using qRT-PCR, Western Blot and immune fluorescence in GS-2 patient-derived MSCs and iPSCs. gRNAs and donor DNAs were designed, based on the patient mutations in exon 3 and exon 7 using the CHOPCHOP online tool and transfected into GS-2 MSCs and iPSCs by electroporation. Cells were cultured for two days and then used for mutation analysis using DNA sequencing.ResultsMSCs and iPSCs from the GS-2 patients lacked expression of both RAB27A gene and protein expression. After gRNA and donor DNAs were designed and optimized, we found an 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.ConclusionUse 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.

Author ORCID Identifier

ÖZGÜR EROL: 0000-0001-9301-5401

ŞİMAL ŞENOCAK: 0000-0002-4030-4215

BURCU ÖZÇİMEN: 0000-0001-7623-8662

GÜLEN GÜNEY ESKEN: 0000-0002-3158-1587

HASAN KILIÇ: 0000-0002-3204-5567

YUSUF KOCAEFE: 0000-0003-3216-9399

NIEK VAN TIL: 0000-0002-9515-4103

FATIMA AERTS KAYA: 0000-0002-9583-8572

DOI

10.55730/1300-0152.2705

Keywords

CRISPR/Cas9, Electroporation, Homology directed repair, non-homologous end joining

First Page

290

Last Page

298

Creative Commons License

Creative Commons Attribution 4.0 International License
This work is licensed under a Creative Commons Attribution 4.0 International License.

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