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. 2025 Jun;29(3):349-357.
doi: 10.18699/vjgb-25-38.

Generation of the ICGi019-B-1 and ICGi019-B-2 lines via correction of the p.Met659Ile (c.1977G>A) variant in MYH7 of patient-specific induced pluripotent stem cells using CRISPR/Cas9

A E Shulgina  1 , S V Pavlova  1 , J M Minina  1 , S M Zakian  1 , E V Dementyeva  1
Affiliations

Generation of the ICGi019-B-1 and ICGi019-B-2 lines via correction of the p.Met659Ile (c.1977G>A) variant in MYH7 of patient-specific induced pluripotent stem cells using CRISPR/Cas9

A E Shulgina et al. Vavilovskii Zhurnal Genet Selektsii. 2025 Jun.

Abstract
in English, Russian

The problem of interpretation of the genetic data from patients with inherited cardiovascular diseases still remains relevant. To date, the clinical significance of approximately 40 % of variants in genes associated with inherited cardiovascular diseases is uncertain, which requires new approaches to the assessment of their pathogenetic contribution. A combination of the induced pluripotent stem cell (iPSC) technology and editing the iPSC genome with CRISPR/Cas9 is thought to be the most promising tool for clarifying variant pathogenicity. A variant of unknown significance in MYH7, p.Met659Ile (c.1977G>A), was previously identified in several genetic screenings of hypertrophic cardiomyopathy patients. In this study, the single nucleotide substitution was corrected with CRISPR/Cas9 in iPSCs generated from a carrier of the variant. As a result, two iPSC lines (ICGi019-B-1 and ICGi019-B-2) were generated and characterized using a standard set of methods. The iPSC lines with the corrected p.Met659Ile (c.1977G>A) variant in MYH7 possessed a morphology characteristic of human pluripotent cells, expressed markers of the pluripotent state (the OCT4, SOX2, NANOG transcription factors and SSEA-4 surface antigen), were able to give rise to derivatives of three germ layers during spontaneous differentiation, and retained a normal karyotype (46,XY). No CRISPR/Cas9 off-target activity was found in the ICGi019-B-1 and ICGi019-B-2 iPSC lines. The maintenance of the pluripotent state and normal karyotype and the absence of CRISPR/Cas9 off-target activity in the iPSC lines with the corrected p.Met659Ile (c.1977G>A) variant in MYH7 allow using the iPSC lines as an isogenic control for further studies of the variant pathogenicity and its impact on the hypertrophic cardiomyopathy development.

Проблема интерпретации результатов генетического анализа пациентов, страдающих наследственными сердечно-сосудистыми заболеваниями, по-прежнему сохраняет свою актуальность. На сегодняшний день клиническое значение около 40 % вариантов в генах, ассоциированных с наследственными сердечно-сосудистыми заболеваниями, остается неясным, что приводит к необходимости использования новых подходов для оценки патогенетического вклада этих вариантов. Совместное применение технологии индуцированных плюрипотентных стволовых клеток и редактирования их генома с помощью системы CRISPR/ Cas9 считается наиболее перспективным способом выяснения патогенности генетических вариантов. Ранее в нескольких генетических скринингах пациентов с гипертрофической кардиомиопатией был выявлен вариант с неясным клиническим значением в гене MYH7, p.Met659Ile (c.1977G>A). В настоящем исследовании данная однонуклеотидная замена с помощью системы CRISPR/Cas9 была исправлена в индуцированных плюрипотентных стволовых клетках, полученных от носителя этого генетического варианта. В результате получены и охарактеризованы с использованием стандартного набора методов две линии индуцированных плюрипотентных стволовых клеток (ICGi019-B-1 и ICGi019-B-2). Линии индуцированных плюрипотентных стволовых клеток с исправленным вариантом p.Met659Ile (c.1977G>A) в гене MYH7 имели характерную для плюрипотентных клеток человека морфологию, экспрессировали маркеры плюрипотентного состояния (транскрипционные факторы OCT4, SOX2, NANOG и поверхностный антиген SSEA-4), были способны давать производные трех зародышевых листков при спонтанной дифференцировке и сохраняли нормальный кариотип (46,XY). В линиях индуцированных плюрипотентных стволовых клеток ICGi019-B-1 и ICGi019-B-2 не обнаружено нецелевой активности системы CRISPR/Cas9. Поддержание плюрипотентного состояния и нормального кариотипа, а также отсутствие нецелевой активности системы CRISPR/Cas9 в линиях индуцированных плюрипотентных стволовых клеток с исправленным вариантом p.Met659Ile (c.1977G>A) в гене MYH7 позволят использовать полученные линии в качестве изогенного контроля для дальнейших исследований патогенности данного генетического варианта и его влияния на развитие гипертрофической кардиомиопатии.

Keywords: CRISPR/Cas9; hypertrophic cardiomyopathy; induced pluripotent stem cells; variants of unknown significance.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Table 1.
Table 1.. Oligonucleotides and antibodies used in this study
Fig. 1.
Fig. 1.. Correction of the p.Met659Ile (c.1977G>A) variant in MYH7 of the patient-specific iPSCs using CRISPR/Cas9.
a – design of single-guide RNA and single-stranded donor oligonucleotide for MYH7 editing. The nucleotide sequence of a fragment of intron 17 and exon 18 is given. The positions of the protospacer for the single-guide RNA, PAM, and the single-stranded donor oligonucleotide are indicated in grey, magenta, and yellow, respectively. The target substitution and synonymous substitution in PAM are shown with red rectangles; b – an example of two iPSC clones with the corrected p.Met659Ile (c.1977G>A) variant in MYH7 (ICGi019-B-1 and ICGi019-B-2). Nucleotide sequences of the same region in the patient-specific iPSC line (ICGi019-B) and the iPSC line of the healthy donor (ICGi022-A) are provided for comparison. The target substitution and synonymous substitution in PAM are shown with red rectangles; c – absence of CRISPR/Cas9 off-target activity at one of the predicted CRISPR/Cas9 off-target sites in the iPSC lines with the corrected p.Met659Ile (c.1977G>A) variant in MYH7 (ICGi019-B-1 and ICGi019-B-2). The nucleotide sequence of the same region in the patient-specific iPSC line used for MYH7 editing (ICGi019-B) is given for comparison. The CRISPR/Cas9 off-target site and its positions in the human genome (hg38) are indicated in red.
Fig. 2.
Fig. 2.. Characterization of the iPSC lines with the corrected p.Met659Ile (c.1977G>A) variant in MYH7.
a – karyotype of the iPSC lines with the corrected p.Met659Ile (c.1977G>A) variant in MYH7 (ICGi019-B-1 and ICGi019-B-2); b – morphology of the ICGi019-B-1 and ICGi019-B-2 iPSC lines. Scale bar 250 μm; c – expression of the OCT4 and SOX2 transcription factors and SSEA-4 surface antigen in the ICGi019-B-1 and ICGi019-B-2 iPSC lines. Scale bar 100 μm; d – expression of pluripotency genes, NANOG and SOX2, in the ICGi019-B-1 and ICGi019-B-2 iPSC lines in comparison with the original patient-specific ICGi019-B line. Data are presented as mean ± SEM; e – capacity of the ICGi019-B-1 and ICGi019-B-2 iPSC lines to be differentiated into derivatives of three germ layers: ectoderm (TUBB3, β3- tubulin), mesoderm (αSMA, smooth muscle α-actin), and endoderm (CK18, cytokeratin 18). Scale bar 100 μm; f – absence of mycoplasma contamination in the ICGi019-B-1 and ICGi019-B-2 iPSC lines. Ctr+, positive control for mycoplasma contamination, H2O, negative control for mycoplasma contamination
Table 2.
Table 2.. Passport of the ICGi019-B-1 and ICGi019-B-2 iPSC lines
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