Generation and genetic repair of two human induced pluripotent cell lines from patients with Epidermolysis Bullosa simplex and dilated cardiomyopathy associated with a heterozygous mutation in the translation initiation codon of KLHL24

Fibroblasts from two patients carrying a heterozygous mutation in the translation initiation codon (c.2 T > G) of the kelch-like protein 24 (KLHL24) gene were used to generate human induced pluripotent stem cells (hiPSCs), using non-integrating Sendai virus to deliver reprogramming factors. CRISPR-Cas9 editing was used for genetic correction of the mutation in the patient-hiPSCs. The top-predicted off-target sites were not altered. Patient and isogenic hiPSCs showed typical morphology, expressed pluripotency-associated markers, had the capacity for in vitro differentiation into the three germ layers and displayed a normal karyotype. These isogenic pairs will enable in vitro modelling of KLHL24-associated heart and skin conditions.


Resource Table:
Unique stem cell lines identifier

Resource utility
Patients with mutations in the translation initiation codon of KLHL24 display Epidermolysis Bullosa Simplex (EBS) associated with high risk of developing Dilated Cardiomyopathy (Has et al., 2020). hiPSCs were generated from two patients with heterozygous c.2 T > G mutation and CRISPR-Cas9 was used to correct the mutation, providing isogenic pairs for in vitro disease modelling.

Resource details
Initially identified as causing genetically unresolved EBS, monoallelic mutations in the translation initiation codon of KLHL24 were recently associated with the development of dilated cardiomyopathies (Has et al., 2020). The encoded protein, KLHL24, belongs to the ubiquitin-proteasome system that regulates protein turnover; however, the pathological mechanisms that cause the disease phenotype remain poorly defined. The generation of mutant and genetically corrected isogenic hiPSC lines offers an unlimited source of the different cell types and enables the study the KLHL24-associated pathologies in a wellcontrolled disease model. Fibroblasts were isolated from skin biopsies of two male patients with a heterozygous c.2 T > G mutation in KLHL24 and were reprogrammed at passage 3 into hiPSCs using replicationdefective and persistent Sendai virus carrying OCT3/4, SOX2, KLF4, MYC (Nishimura et al., 2011). One hiPSC line was characterized for each patient: lines LUMCi045-A and LUMCi046-A (Table 1). LUMCi045-A and LUMCi046-A were negative for SeV after 7 passages as indicated by qRT-PCR ( Supplementary Fig. 1A). They displayed a typical stem cell morphology with high nucleus to cytoplasm ratio (Fig. 1A) and a normal karyotype as assessed by G-banding (passages 22) and KaryoStat assay (passage 15 and 22, respectively) ( Fig. 1F,G). Their pluripotency status was confirmed by expression of pluripotency markers: SSEA4, NANOG and OCT3/4 were clearly detected by immunofluorescence staining (Fig. 1C) and SSEA4 and NANOG were found by flow-cytometry (Fig. 1D). The lines were able to differentiate into derivatives of all three germ layers in a 'spontaneous differentiation' assay in vitro, as illustrated by immunofluorescence staining for the ectodermal marker βIII-tubulin (B3-TUB), the endodermal marker α-fetoprotein (AFP) and the mesodermal marker platelet endothelial cell adhesion molecule-1 (PECAM-1) (Fig. 1E). The presence of heterozygous c.2 T > G mutation in exon 4 of KLHL24 was confirmed by Sanger sequencing (Fig. 1B). The mutated allele was repaired by a Cas9-ribonucleoprotein (RNP) complex, with a mutation-specific single guide RNA (sgRNA) and a single-stranded oligodeoxynucleotide (ssODN) donor template containing the wild-type sequence with a silent mutation either to introduce a AvaII restriction site or to disrupt the MaeIII site (ssODN-1 and − 2, for LUMCi045-A-1 and LUMCi046-A-1, respectively; Table 1). Single-cell derived colonies were screened for repair by using the AvaII (for LUMCi045-A-1) or MaeIII (for LUMCi046-A-1) restriction of PCR amplified KLH24 start codon region. Correction of the mutation was confirmed by Sanger sequencing (Fig. 1B). The repaired hiPSCs showed the expected morphology and a normal karyotype (passage 6 after gene editing) using G-banding (Fig. 1A,G). Immunofluorescence staining and flow-cytometry analyses revealed expression of the pluripotency markers ( Fig. 1C,D) and spontaneous differentiation into the three germ layers was observed in vitro, as illustrated by immunofluorescence staining with specific markers as described above (Fig. 1E). All lines were mycoplasma negative ( Supplementary Fig. 1B). The origin of the isogenic pairs was confirmed by short tandem repeat (STR) analysis which fully matched the profile of the patient's fibroblasts. Finally, the absence of off-target mutations was confirmed by Sanger sequencing of the top5 as well as all the exon sites predicted by CRISPOR (crispor.tefor. net; data not shown) (Haeussler et al., 2016). The complete characterization is summarized in Table 1.

Gene editing
10 5 cells (LUMCi045-A and LUMCi046-A, passages 15 and 28) were transfected with the Cas9-RNP complex and the ssODN (Integrated DNA Technologies) using the NEON Transfection System (Invitrogen) at 1200 V/30 ms/1pulse. Cells were plated on Corning® Synthemax® II-SC substrate (Merck, # CLS3535-1EA) using TeSR TM -E8 TM with CloneR (Stemcell Technologies, #05888). After recovery, 1000 cells were plated on Synthemax II-SC-coated 10 cm dish in TeSR TM -E8 TM with CloneR and single cell-derived colonies were screened for the corrected genotype. DNA was isolated using QuickExtract TM DNA Extraction Solution (Lucigen, #QE0905T) and the region of interest was amplified by PCR (Terra PCR Direct Polymerase Mix, TaKaRa; Bio-Rad S1000 Thermal Cycler; program available upon request). Corrected clones were identified by analysis of the restriction pattern obtained after digestion of the PCR fragment with AvaII (LUMCi045-A-1) or MaeIII (LUMCi046-A-1) (New England Biolabs) and confirmed by sequencing.

Sequencing
Sanger sequencing was performed by Leiden Genome Technology Centre using the ABI3730xl system.

Immunofluorescence staining
Immunofluorescence staining and imaging were performed as described (Bouma et al., 2019). Primary and secondary antibodies are reported in Table 2.

Flow-cytometry analysis
Cells were dissociated into single cells with Gentle Cell dissociation reagent (Stemcell Technologies, #07174) and fixed and permeabilized using the FIX & PERM TM Cell Permeabilization kit (ThermoFisher, #GAS004). Cells were incubated with the antibodies (Table 2) for 1 h in the dark at RT and analyzed with a LSRII flow-cytometer (BD). The HACAT keratinocyte line was used as a negative control.

Karyotyping
G-banding analysis was conducted at the Laboratory of Clinical Genetics Leiden (LDGA). A total of 20 metaphases was analyzed. The KaryoStat assay was performed according to manufacturer's instructions (ThermoFisher Scientific).

Evaluation of off-target effects
The Top5 and exonic off-target sites were predicted using the CRISPOR online tool (crispo.tefor.net) (Haeussler al., 2016). PCR products of the predicted sites were analyzed by sequencing.

Mycoplasma detection
The mycoplasma status was assessed using the MycoAlert TM mycoplasma detection kit (Lonza, #LT07-418) following the manufacturer's protocol.

STR analysis
Cell line authentication was performed by the Department of Human Genetics, LUMC, by using the PowerPlex® Fusion System 5C autosomal STR kit (Promega) as previously described (Westen et al., 2014).

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.