EFFICIENCY OF POINT MUTATION BY CRISPR/ CAS9 IN IPSCS DETERMINED BY NEXT GENERATION SEQUENCING

GUADALUPE AMIN; SHEILA LUCIA CASTAÑEDA; ALAN MIQUEAS MÖBBS; MARIA AGUSTINA SCARAFIA; ALEJANDRO DAMIÁN LA GRECA; ARIEL WAISMAN; CATALINA ATORRAGASTI; SANTIAGO GABRIEL MIRIUKA; LUCIA NATALIA MORO

Mar del Plata

Congreso; LXVI Reunion Anual de Sociedades de Biociencias; 2021

Sociedad Argentina de Investigacion Clinica (SAIC)

The arrhythmogenic cardiomyopathy (ACM) is a genetic disease characterized by the replacement of contractile myocardium by adipose tissue, causing ventricular arrhythmias and eventually sudden death in patients. One of the most commonly mutated genes is the Plakophilin-2 (PKP2) that codifies for a desmosomal protein. The aim of this work was to generate an induced pluripotent stem cell (iPSCs) line with a reported point mutation in PKP2 gene (C>T that generates a missense mutation p.S140F) by CRISPR/Cas9 for modeling the ACM in vitro. In order to generate this edition we designed two RNA guides (gRNA 1 and 2) targeting the Cas9 to the desired region of the PKP2 gene and a template DNA for each gRNA (ssODN 1 and 2) complementary to the sequence containing the point edition. We co-transfected the plasmid containing the CRISPR system with gRNA1 or gRNA2 together with the ssODN1 or ssODN2, respectively, to 2x10⁵ iPSCs in two different concentrations (1 ug or 2.5 ug of each DNA construction): gRNA1-1, gRNA1-2.5, gRNA2-1, gRNA2-2.5 groups. After puromycin selection, genomic samples from the 4 groups were taken for amplicon sequencing analysis. PCR amplicons from the pool were sequenced using Miseq in CD-Genomics. These results were analysed with CRIS.py, a python-based program for multiple sequence analysis. The analysis revealed 24.7%, 27.6%, 0.2% and 5.5% of C>T edition, 9%, 10.9%, 3.2%, 25.7% of indel, and 67.8%, 62.9%, 91.2% and 47% of wild type sequences for gRNA1-1, gRNA1-2.5, gRNA2-1, gRNA2-2.5, respectively. With these results, cells from gRNA1-1 were clonally expanded and 15 clonal cell lines were Sanger sequenced, obtaining 3 clonal cell lines with the desired edition (20%). In conclusion, gRNA1 was more efficient than gRNA2 independently of the DNA concentration used. Our next steps are to characterize the phenotype of the C>T PKP2 clonal cell lines and to determine whether we can model the ACM in-vitro after differentiating these cell lines into cardiomyocytes.