Perinatal death in pig models of hypertrophic cardiomyopathy carrying sarcomere pathogenic variants

Figures

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  Fig. 1. Gene disruption strategy in exon 6 and exon 23 of porcine MYBPC3. The CRISPR/Cas9 genome editing was performed with non-homologous end-joining to introduce insertion and/or deletion in the MYBPC3 gene in primary porcine kidney fibroblasts. A) structure of the porcine MYBPC3 gene and localisation of the PAM sequences (light green) and sgRNA (pink) for both exon 6 and exon 23. B) Examples of nucleotide sequence alignment by Sanger sequence analysis of the sgRNA targeted region in single cell clones. Deletion and insertion are indicated by dash and red bold letters, respectively. Numbers indicate insertion (+) or deletion (−) created via CRISPR/Cas9. Amino acid sequences of the targeted regions are shown above the nucleotide sequences (new amino acids in blue and new PTC marked with Asterix in red). Exon 24 (partial) is underlined. Abbreviations: PAM, protospacer adjacent motif: PTC, premature termination codon; sgRNA, single guide RNA. Figure created with BioRender.com. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 2. Analysis of the genotype and phenotype of the stillborn MYBPC3 mutant piglet. A) Agarose gel showing the PCR amplicons from genomic DNA in the stillborn piglet. The expected 812-bp fragment and a 1.2-kb fragment were amplified; 1, ear clip; 2, kidney cells; +wt, wild-type PKF; -H2O, water. B) Structure of the mutant MYBPC3 alleles. Top panel (allele 1): A partial sequence of the WT and mutant exon 23 is shown, including the PAM sequence (green) and a 1-bp (cytosine) deletion (dash). The 1-bp deletion in exon 23 causes a frameshift, resulting in an amino acid exchange (pink) and a PTC (asterisk) in exon 24 (underlined sequence, partial). Bottom panel (allele 2): A 361-bp insertion is highlighted in brown. A partial sequence shows the insertion that includes 10 bp of exon 22, 289 bp of intron 22 (with the splice donor and acceptor sites highlighted in bold), and 62 bp of exon 23. The insertion leads to the duplication of 24 amino acids between positions 779 and 802 (green) of the WT MYBPC3 protein. C) RT-qPCR analysis for MYBPC3, NPPA and NPPB performed on RNA extracted from WT myocardial tissue (Heart) of pigs (n = 2, 7-day-old male and 2-month-old female pigs) and from left…

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  Fig. 3. MYH7 base editing and analysis of the genotype and phenotype of stillborn MYH7 mutant piglets. The CRISPR/Cas9 base-editing was used to introduce the c.1357C>T transition in the MYH7 gene in primary Landrace porcine fibroblasts. A) Structure of the porcine MYH7 gene and localisation of the PAM sequences (light green) and sgRNA (pink) in coding exon 12. The targeted cytosine is depicted in bold. B) PCR on porcine fibroblast genomic DNA, followed by NlaIII digestion of porcine fetal fibroblasts showing the wild-type allele (427-bp fragment) and the digested mutant allele (two fragments of 246 bp and 181 bp). C) Sequencing results of the undigested PCR fragment showing the heterozygous c.1357C>T MYH7 genetic variant in mutant porcine fibroblasts. D) PCR on genomic piglet ear DNA, followed by NlaIII restriction site digestion showing the wild-type allele (427-bp fragment) and the digested mutant allele (two fragments of 246 bp and 181 bp) in two mutant MYH7 piglets (P1 and P2). E) RT-PCR analysis followed by NlaIII digestion performed around exon 12 of MYH7 on RNA extracted from heart of both mutant piglets showing the WT mRNA (125-bp fragment) and the 2 NlaIII-digested fragments (79…