Generation of eight hiPSCs lines from two pathogenic variants in CACNA1A using the CRISPR-Cas9 gene editing technology

Developmental and epileptic encephalopathies (DEEs) are rare severe neurodevelopmental disorders with a cumulative incidence of 1:6.000 live births. Many epileptic conditions arise from single nucleotide variants in CACNA1A (calcium voltage-gated channel subunit alpha1 A), encoding the CaV2.


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Many epilepsy-associated genes have been identified; however, the underlying molecular pathomechanisms and cell type specific disease phenotypes remain largely unknown.To better understand disease development and the underlying molecular mechanisms on cellular levels, we established eight hiPSC lines containing two pathogenic variants (either heterozygous or homozygous) in CACNA1A.

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Epilepsy is a frequent symptom observed in patients with de novo pathogenic CACNA1A variants and most of the de novo missense variants cluster in the voltage sensor and the pore of the channel (Yu et al., 2014).Different epilepsies rely on different functional mutant channel properties (Oyrer et al., 2018;Maguire, 2021).Here we generated a hiPSC lines for disease modelling DEE by introducing specific variants in CACNA1A, previously described in epileptic patients, via CRISPR-Cas9 gene editing technology (Jinek et al., 2012).We introduced the c.2139 G > A variant which is predicted to result in an amino acid change from alanine (A) to threonine (T) (A713T) and the c.1767C > T variant predicted to result in an amino acid change from arginine (R) to cysteine (C) (R589C) in CACNA1A in the human iPSC line BIONi010-C.This cell line had earlier been established from a skin biopsy obtained from a healthy male individual aged 18 (Rasmussen et al., 2014).
Eight lines were generated using CRISPR-Cas9 gene editing technology, which are referred to as A713T and R589C, respectively, in the figures.The nucleotide substitution was confirmed by restriction enzyme digestion (data not shown) and Sanger sequencing.Sequencing analysis of the eight lines is illustrated in (Fig. 1A).
Pluripotency of the gene-edited lines was confirmed by immunocytochemistry (ICC) and quantified by quantitative reverse transcription PCR (RT-qPCR).All cell lines showed clear expression of OCT4, NANOG, SSEA4, SSEA3, TRA-1-60 and TRA-1-81 via ICC (Fig. 1B).Expression of the pluripotency genes (OCT4, SOX2, NANOG) was demonstrated by RT-qPCR and the normalization of gene expression levels were validated with a normal dermal fibroblast cell line (Fig. 1C).All cells-lines were investigated by light microscopy (Fig. 1D), confirming a normal hiPSC morphology throughout the gene editing process.The differentiation potential of the hiPSCs was confirmed via spontaneous differentiation into endoderm, ectoderm, and mesoderm cell types with ICC (Fig. 1E).Large chromosomal rearrangements were tested using Applied Bio-systems™ Cytoscan™ 750 K arrays using filters detecting microdeletions or microduplications above 500 kb.No large chromosomal aberrations were observed; however, we did observe the same microduplication in the parental cell line (Table 1) at Chr22q11.2 as described in EBiSC (https://ebisc.org/BIONi010-C)(Supplemental Fig. 1A, B).Finally, short tandem repeat (STR) analysis confirmed cell line identity (available upon request) and all hiPSCs were tested negative for mycoplasma (Supplemental Fig. 1C).

Gene editing
Cells were cultured on Matrigel coated plates supplemented with Essential 8 (E8) media and incubated at 37 • C and 5% CO 2 .After reaching 80% confluency, cells were detached with Accutase for 5 min after which 1x10 6 cells were nucleofected using the Lonza nucleofector, P3 solution kit with the CA167 pulse setting.Nucleofected cells were plated on Matrigel coated plates, and colonies were picked manually after 7 days for analysis.Gene editing was performed implementing the CRISPR-Cas9 gene editing technology.To introduce the desired point mutations in CACNA1A, two sgRNA sequences were designed together with a ssODN (single stranded oligodeoxynucleotide) as homologous template containing the specific mutation and a silent mutation (design information available in Table 2).The silent mutation (that changes the DNA sequence but not the protein) was inserted to avoid repeated cutting by Cas9.Mutation specific restriction enzyme Tsp45I and HphI were used to recognize the specific mutation sites for Ala713Thr and Arg589Cys, respectively.The sgRNA was designed using the software http://crispr.mit.edu/, and the ssODNs were designed to include "base pairs of interest".ssODNs and Cas9 protein were ordered from Integrated DNA Technologies (IDT), and the sgRNAs were ordered from Synthego.

Genotyping and restriction digest
To analyze the hiPSC clones, DNA extraction was performed with the prepGEM kit (ZyGEM), followed by DNA amplification with the AmpliTaq GOLD DNA polymerase (ThermoFisher) according to the manufacturer's instructions.The primers used for PCR amplification  were designed to span the CACNA1A variants (detailed in Table 2).The screening of Ala713Thr was carried out by restriction enzyme digestion of the PCR product using Tsp45I for 1 hr at 65 • C, and for Arg589Cys with HphI for 1 hr at 37 • C (New England Biolabs).For each mutation we picked 192 clones and processed those for identifying the positive clones.Out of the 192 clones, 30-40 clones were selected for Sanger sequencing using the corresponding primers for respective variants (Table 2).Sequence-verified clones were then expanded from a single colony to ensure the purity of the population.

Quality control
Quality control (detailed in Table 2) was performed.The overall morphology of the cells was assessed daily by light microscopy.Genomic integrity quality check was carried out at passage (P) 25 (5 passages after nucleofection) for the cells carrying the variants Ala713Thr and Arg589Cys.When the cells reached 70-85% confluency, DNA were extracted using the DNeasy Blood & Tissue kit from Qiagen and tested for large chromosomal rearrangements using Cytoscan™ 750 K arrays Department of Clinical Genetics, Odense Universitetshospital, Denmark.STR analysis was performed using the AmpFLSTR Identifier PCR Amplification kit according to manufacturer's instructions (Applied Biosystems).Mycoplasma test was carried out with mycoplasma test kit (VenorGeM) according to the manufacturer's instruction.Briefly, the culture medium of hiPSC with 80% confluence, at P30 was collected (100 μl), heated for 5 min at 95 • C, and centrifuged for 2 min at maximum speed.Positive and negative controls were also set up for a standard PCR to amplify the 16S rRNA of the genus Mycoplasma with the following program: 95 • C 2 min; 5 cycles: 94 • C 30 sec, 50 • C 30 sec, 72 • C 35 sec; 30 cycles: 94 • C 15 sec, 56 • C 15 sec, 72 • C 30 sec.

Immunostaining
The hiPSC clones were fixed at P30 with 4% paraformaldehyde (PFA) for 20 min at room temperature (RT) and labeled according to standard ICC procedures by permeabilizing fixed cells with 0.2% Triton X-100 in PBS for 20 min at RT followed by 30 min of blocking with 3% bovine serum albumin (BSA).All primary antibodies were diluted in 3% BSA and incubated overnight at 4 • C (antibody list detailed in Table 2).The following day, primary antibodies were washed, and secondary antibodies were added for one hour RT.The cells were washed and then stained with HOECHST 33342 (nuclei).Finally, the cells were mounted in DAKO S3023 media and the samples were analyzed in a Leica DMRBfluorescence microscope at wavelengths 488 nm and 594 nm.

RNA extraction and RT-qPCR
RT-qPCR was carried out to quantify the expression of pluripotency markers.Cells were collected at P30, after which RNA extraction was performed using RNeasy Plus Mini Kit (QIAGEN).cDNA synthesis was performed with the SuperScript™ IV VILO™ Master Mix with ezD-Nase™ Enzyme (ThermoFisher).RT-qPCR was carried out using the QuantStudio™ 3 Real-Time PCR System (Thermo Fisher) using the comparative CT SYBR® green program.The conditions were: 95 • C 15 s; 60 • C 1 min for 40 cycles; melt curve 95 • C 15 s, 60 • C 1 min, 95 • C 1 s.Expression was quantified using the ΔΔ Ct method with GAPDH as reference.The primers used for RT-qPCR are listed in Table 2.

Spontaneous differentiation
The spontaneous differentiation potential of hiPSCs to the three embryonic germ layers was evaluated using the embryoid body (EB's) method.Cells were cultured in low adherent plates in E8 media for 7 days.After which the formed EB's were spontaneously differentiated by seeded onto Matrigel-coated coverslips and cultured for 14 days in fibroblast media.Differentiated cells were analyzed by immunofluorescence staining for smooth muscle actin (mesoderm), alpha fetoprotein (endoderm), and beta-3-tubulin (ectoderm).Antibodies used for immunofluorescence are listed in Table 2.

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.

Fig. 1 .
Fig. 1.A) Characterization of CACNA1A gene edited hiPSC: Sanger sequencing of CACNA1A showed a heterozygous and a homozygous insertion of the pathogenic variants c.2139G > A, p.(Ala713Thr) (top panel) and c.1767C > T, p.Arg589Cys) (bottom panel) depicted by a blue arrow.The gene edited lines are referred to as A713T and R589C, respectively.In addition, silent variants were inserted (red arrows).B) Characterization of the hiPSC lines derived from induced pluripotent stem cells with the variants A713T (top panel) and R589C (bottom panel).Representative immunofluorescence images of the stem cell markers OCT4 (red), SSEA4 (green), NANOG (red), TRA-1-81 (green), TRA-1-60 (red) and SSEA3 (red), Scale bar = 200um.C) Quantification of NANOG, OCT4 and SOX2 expression by qRT-PCR.CT-values were normalized to the GAPDH and expression was calculated relative to fibroblasts using the ΔΔCT-method.D) Bright-field image of the eight geneedited hiPS cell lines of A713T (left panel) and R589C (right panel) Scale bar = 1000um.E) Representative immunofluorescence images of endoderm, mesoderm, and ectodermal cell types with the respective markers AFP, SMA and β-tubulin III, A713T (top panel) and R589C (bottom panel), Scale bar = 200um.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 1
Characterization and validation.