Targeted correction of megabase-scale CNTN6 duplication in induced pluripotent stem cells and impacts on gene expression

Human iPS cells culture conditions and electroporation

Human iPS cells were previously generated by reprogramming skin fibroblasts (Gridina et al., 2018). iPS cells were cultured and expanded under feeder-free conditions, using TeSR-E8 medium (Stemcell Technologies, Vancouver, Canada) in dishes coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer’s recommendations.

Electroporation was utilized to insert loxP sites at the targeted locations within the second copy of the CNTN6 gene in human iPS cells. The cells were cultured in TeSR-E8 medium, supplemented with 1% Penicillin-Streptomycin (Pen-Strep) on Matrigel-coated dishes (according to the manufacturer’s manual). The iPS cells were seeded in a 6-well plate (30 × 10³ cells per cm²). Electroporation was performed when 70% confluency was reached. One hour before electroporation, the medium was switched to TeSR-E8 without antibiotics, but with the addition of 10 µM ROCK inhibitor. The cells were treated with TrypLE solution, rinsed with DMEM/F12 medium containing 10% KnockOut Serum Replacement, and then washed with phosphate buffered saline (PBS). After removing the supernatant thoroughly, the cells were resuspended in Buffer R (Neon^™ electroporation system Kit) at a concentration of 180 × 10³ cells/µl. Transfection was carried out using the Neon^™ electroporation system in a 10 µl volume under the following conditions: 1,200 V and 30 µs per pulse. The iPS cells were transfected with a combination of 2 µM single-stranded oligodeoxynucleotides (ssODN) and 0.5 µg of the pSpCas9(BB)-2A-Puro (PX459) V2.0 plasmid (#62988; Addgene, Watertown, MA, USA) or 1 µg of the Cre-IRES-PuroR plasmid (#30205; Addgene, Watertown, MA, USA). Guide RNA and ssODN sequences were designed using the online resource Benchling (https://www.benchling.com). Post-electroporation, the cells were cultured on Matrigel in TeSR-E8 medium with ROCK inhibitor and without antibiotics. Twenty-four hours after electroporation, 0.4 µg/ml puromycin was added to the culture medium for selection, which was conducted over the next 3 days. Ten days later, emerging clones were manually transferred to 24-well plates.

Droplet digital polymerase chain reaction

Droplet digital polymerase chain reaction (ddPCR) was conducted using a Bio-Rad QX100 system, adhering to the manufacturer’s guidelines. The reaction volume was 20 µl, consisting of 1× ddPCR^™ Supermix for Probes (no dUTP), 900 nM primers, 250 nM probes, and the DNA template. The sequences for primers and probes are provided in Table S1. Each sample’s ddPCR reactions were performed in duplicate. Droplets were generated using DG8^™ cartridges and transferred to PCR plates, then analyzed using a droplet reader with Quanta Soft software (Bio-Rad, Hercules, CA, USA). The procedure was replicated for three independent biological replicates. The human ACTB gene was used as the reference gene. The absolute number of CNTN6 complementary DNA (cDNA) copies obtained via ddPCR was normalized to the cDNA copies of the ACTB gene.

Statistical analysis

All statistically analyzed data followed a normal distribution, as confirmed by the Shapiro-Wilk test. The statistical significance was then assessed using a Student’s t-test.

Ngn2-induced differentiation of iPS cells to iN cells

Induced neuronal (iN) cells were generated from iPS cells via the forced expression of the neurogenin-2 transcription factor (encoded by the Ngn2 gene), as previously described (Gridina et al., 2018). In brief, we utilized lentiviral constructs: FUW-TRE Ngn2/Puro, containing full-length mouse Ngn2 and puromycin-resistance genes, and FUW-TRE EGFP, containing Green Fluorescent Protein (GFP). Lentiviruses were produced in the Phoenix cell line using Lipofectamine 3000 (Invitrogen, Waltham, MA, USA), following the manufacturer’s recommendations. The iPS cells were transduced with these lentiviruses in a culture medium supplemented with 5 µg/ml polybrene (MilliporeSigma, Burlington, MA, USA). To initiate Ngn2 gene expression, 2 µg/ml doxycycline (DOX; Sigma-Aldrich, St. Louis, MI, USA) was added to the medium 1 day post-transduction, which consisted of DMEM/F12, 1% MEM NEAA, 1% Pen-Strep, 1% N2 supplement, human brain derived neurotrophic factor (BDNF) (10 ng/ml), mouse laminin (0.2 µg/ml; Sigma-Aldrich, St. Louis, MI, USA), and human Neurotrophin-3 (NT-3) (10 ng/ml; PeproTech, Cranbury, NJ, USA). The Ngn2-expressing cells were selected using 1 µg/ml puromycin (Gibco, Waltham, MA, USA) starting the day after DOX induction. Following 1 day of puromycin selection, the cells (45 × 103 cells/cm2) were plated on mitomycin C-treated mouse glial cells. The cultures were then maintained for 3 weeks in Neurobasal Medium supplemented with 2% B27, 1% GlutaMAX^™-I, 1% Pen-Strep, human BDNF (10 ng/ml), and NT-3 (10 ng/ml, all from Gibco, Waltham, MA, USA). Total RNA was subsequently extracted from the iN cells for droplet digital PCR analysis.

Insertion of loxP sites flanking a single copy of the duplicated CNTN6 gene in iPS cells

To remove one copy of the duplicated CNTN6 gene in iPS cells, we utilized a combination of CRISPR/Cas9-mediated genome editing and the Cre/loxP system. The experimental design for the deletion is outlined in Fig. 1A. The initial step involves inserting loxP sites at the boundaries of the duplicated segments. Guide RNA (gRNA) target sites were selected 300 bp before the end of the duplicated region (Table S1). Consequently, there are three target breaks in the genome of iPS cells carrying the duplication. The first is at the end of the first duplicated copy (marked as “1”), the second is associated with the second duplication copy (“2”), and the third is on the wild-type CNTN6 allele (“3”). A single-stranded oligodeoxynucleotide (ssODN) was engineered to include homology arms, a loxP site, and a HindIII restriction enzyme site (Table S1). The iPS cells iTAFdup22 were obtained from the skin fibroblast of patients with CNTN6 duplication (Gridina et al., 2018). We electroporated iTAFdup22 cells by the pSpCas9(BB)-2A-Puro plasmid (#62988, Addgene, Watertown, MA, USA), which contains the gRNA, Cas9 gene, and puromycin resistance gene, along with the ssODN. As a result, we obtained 68 clones, designated iTAFdup22-L1-1 through iTAFdup22-L1-68.

We genotyped iPS cell clones using HindIII digestion of PCR products obtained with two sets of primers (Table S1): “1” for detecting loxP site insertion between two duplicated copies and “2,3” for detecting loxP site insertion at the end of the duplicated region, as well as in the wild-type allele (Fig. 1A). The analysis revealed that the obtained clones could be categorized as follows (Table 1):

• 1)

  35 clones without any insertions;

• 2)

  Clones where the double-strand break repair in region #1 likely occurred via non-homologous end joining (NHEJ), resulting in the loss of the junction between duplicated copies (Fig. 1B, clone 67), while having loxP site insertions in both regions #2 and #3;

• 3)

  Clones where double-strand break repair likely occurred via NHEJ in regions #1, #2, and #3 (Fig. 1B, clones 50, 61);

• 4)

  Clones without an insertion in region #1, but with loxP site insertions in both regions #2 and #3 (Fig. 1B, clone 27);

• 5)

  Clones without an insertion in region #1, but with loxP site insertions in either region #2 or #3 (Fig. 1B, clone 48);

• 6)

  Clones with a loxP site insertion in region #1 and either region #2 or #3 (Fig. 1B, clone 66);

• 7)

  Nine clones with loxP site insertions in all three regions (Fig. 1B, clone 6).

The correct insertion of full-length loxP sites was confirmed by Sanger sequencing (Fig. 1C). The selected clone was subsequently subcloned to minimize potential heterogeneity within the cell population.

Excising a region flanked by loxP sites

A subclone was chosen for further manipulation, and the cells were transfected with the Cre-IRES-PuroR plasmid (#30205; Addgene), which contains the Cre recombinase. As a result, 11 subclones were obtained, labeled iTAFdup-cre1 through iTAFdup-cre11. Analysis of these clones revealed that two lacked PCR products corresponding to the junction of the duplicated copies (Figs. 2A, 2B).

To verify the correction of chromosomal rearrangements, we analyzed the ratio of parental alleles of the CNTN6 gene within the duplicated region. The sixth exon of the CNTN6 gene contains single-nucleotide polymorphisms (SNPs) that discriminate the parental alleles (Gridina et al., 2018). DdPCR was conducted on DNA samples isolated from iTAFdup22-cre2 and iTAFdup-cre3, as well as iTAFdup22 (parental iPS cells with the duplication), and iTAF1nor36 (iPS cells derived from healthy donor fibroblasts lacking the duplication). The results demonstrated that after correcting the chromosomal rearrangements, the ratio of parental alleles was nearly 1:1, 0.987 and 0.998 in iTAFdup-cre2 and iTAFdup-cre3 clones, respectively. This ratio matched that of iTAF1nor36 cells from a healthy donor. Conversely, parental iTAFdup22 cells with duplication exhibited an allele ratio of 2:1 (Fig. 2C). The fact that exon 6 of the CNTN6 gene is located at a distance of 167 kb from the introduced CRISPR/Cas9 break, allows us to be confident that the obtained results are not a consequence of damage to the primer landing site. Thus, the chromosomal correction successfully normalized the copy number of the CNTN6 gene in the corrected cell lines.

CNTN6 expression in Ngn2-induced neurons

Previously we demonstrated that CNTN6 expression in neurons carrying the duplication was significantly reduced compared with expression in neurons from healthy donors. Moreover, the expression from the exactly duplicated allele was markedly lower compared to the wild-type allele (Gridina et al., 2018). To assess impacts of duplication correction on CNTN6 gene expression levels, we examined iN cells derived from three different sources: healthy donor iPS cells (iN1nor36), patient iPS cells with a duplicated CNTN6 gene (iNdup22), and iPS cells where one copy of the duplicated CNTN6 gene was removed (iNdup-cre2). These cells underwent neuronal differentiation induced by the forced expression of exogenous Ngn2. The results of the quantitative analysis of CNTN6 gene expression across these clones, based on three replicates, are presented in Fig. 2D. We confirmed previous findings that the CNTN6 expression level in iN cells from patients with a gene duplication was lower than that in iN cells from a healthy donor. However, the removal of one duplicated copy in iNdup-cre2 cells did not increase the expression level of the remaining CNTN6 gene copy. This rather disconcerting result may indicate that this duplication may lead to the establishment of some epigenetic marks in early development that are not erased during reprogramming into iPS cells. “Epigenetic memory” can be retained in iPS cells, even though these cells meet all commonly accepted criteria for pluripotency (Lin, Lehle & McCarrey, 2024).