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

Abstract

Background/aim: 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 methods: 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.

Results: 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.

Conclusion: 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.

Keywords: CRISPR/Cas9; electroporation; homology-directed repair; nonhomologous end joining.

Figure 1

Morphology of healthy donor and GS-2 patient-derived MSCs and iPSCs taken with a light microscope of donor and GS-2 MSC cultures (upper lane) and their iPSCs (lower lane). MSCs show typical fibroblast shapes, whereas iPSCs grow in distinct colonies, as shown in light microscope pictures from representative cultures.

Figure 2

RAB27A expression by donor and GS-2 MSCs and iPSCs. Donor and GS-2 patient-derived MSCs (IK and YF) were stained for RAB27A expression using flowcytometry (upper left) and immunofluorescent staining of adherent cells in culture (upper right, DAPI: blue, RAB27A: red). RAB27A gene expression in GS-2 MSCs from two donors (İK and YF) was calculated relative to RAB27A expression in healthy donor MSCs, whereas GS-2 iPSCs expression of RAB27A was calculated relative to healthy donor iPSCs (lower left). GS-2 iPSC expression of pluripotency genes was confirmed for OCT4, SOX2, and NANOG (lower right).

Figure 3

Genome targeting efficiency of different gRNAs. The genome targeting efficiency of the designed gRNAs (3.2, 3.3, and 3.5 for exon 3 and 7.1, 7.2, and 7.3 for exon 7) was tested using the T7 Endonuclease assay. Upper panel: the DNA of gRNA-transfected cells was controlled with PCR; lower panel: the PCR products were loaded onto a 2% agarose gel to detect gRNA efficiency after adding T7 endonuclease. All tests were run in duplicate. Genome targeting efficiency was the highest with gRNA 3.3 and 7.3 (as shown by the red rectangles in the lower panel).,

Figure 4

Mutation analysis of GS-2 MSCs after transfection. MSCs from two different GS-2 patients (YF: exon 3 delAGinsC; İK: exon 7 delCAAGC) were transfected and cultured for 2 days. Mixed cell populations from a single well were collected for DNA analysis. Sequencing analysis revealed the absence of HDR in exon 3 (A) but the presence of HDR in up to 50% of the cells with exon 7 mutations (B).

Figure 5

Transfection of GS-2 iPSCs with the RAB27A exon 3 mutation (upper left) resulted in loss of viability and spontaneous differentiation (lower left) and low HDR efficacy (right).