Generation of plakoglobin edited pluripotent stem cells by CRISPR/Cas9 for cardiomyopathy modeling

XIMENA GARATE; LUCIA NATALIA MORO; GUADALUPE AMIN; ALEJANDRO DAMIÁN LA GRECA; VERONICA FURMENTO; GABRIEL NEIMAN; NATALIA LUCÍA SANTÍN VELAZQUE; MARIA AGUSTINA SCARAFIA; ALAN MIQUEAS MÖBBS; CARLOS DANIEL LUZZANI; SANTIAGO GABRIEL MIRIUKA

Melbourne

Congreso; International Society for Stem Cell Research; 2018

ISSCR

The arrithmogenic cardiomyopathy (ACM) is a genetic disease that affects mainly the left ventricle due to progressive replacement of myocardium by adipose tissue. Several mutations are reported to cause this cardiac condition, most of them related to desmosomal genes. The aim of this work was to analyze the expression of 5 desmosomal genes (desmoplakin, plakoglobin (Pkg), plakophilin 2 , desmoglein 2 and desmocolin 2 ) during cardiomyocyte differentiation from iPSCs cells and to generate a Pkg knockout iPSCs line by CRISPR/Cas9 in order to model the disease. The differentiation protocol consisted on a monolayer protocol in a serum-free system by temporal modulation of regulators of canonical Wnt signaling. RNA and protein samples were taken on day 0, 3.5, 7 and 21 of differentiation, from 3 independent experiments. We observed cell contractility by day 8, and after qPCR we observed a significant decrease in the expression of pluripotency genes (Oct4 and Nanog) and an increase of the expression of mesoderm-cardiac genes (Btachyury, Nkx25 and cTnT), as the protocol advanced. A tendency to increase the expression of desmosomal genes by day 7 and 21 was also observed, with no statistical differences though. However, a higer expression of PKG was assessed by western blot by day 7 of differentiation. In order to generate an early stop codon in the PKG gene, we designed 2 RNA guides (gRNA1 and gRNA2) to the exon 1 of the gene and a single strand oligo DNA (ssODN) of 70 bp containing the desired mutation and a silent mutation for a restriction site of BamH1. 1 ug of the CRISPR sysem and 1 ug of the ssODN were co-transfected to 200000 iPSCs. After puromycin selection, the cells were clonally espanded and evaluated for the ssODN incorporation. To achieve this, the gene was amplified by PCR and the amplicon was then digested with BamH1. We obtained different efficiencies of ssODN incorporation depending on the gRNA udes: 15.4% (n=2/13) and 71.4% (n=10/14) for the gRNA1 and gRNA2 respectively. In summary, we were able to characterize the expression of the desmosomal genes during the cardiac differentiation protocol and to generate mutated iPSCs lines for Pkg with high efficiency. Now we are planning to differentiate the mutated lines to cardiomyocytes in order to study their phenotype and to determine if we can model the ACM by this strategy.