Generation of a human pluripotent stem cell reporter cell line for the cardiac mesoderm gene MESP1 using CRISPR/Cas9-induced homologous recombination

RODRIGO SKOP; ALAN MIQUEAS MOBBS; JUAN PURACCHIO; SANTIAGO MIRIUKA; ARIEL WAISMAN

Congreso; LVII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research (SAIB); 2021

Heart related diseases are the leading cause of death worldwide. Having appropriate in vitro models to study cardiac differentiation and regeneration is of key importance for the future development of cell therapies. Human pluripotent stem cells (hPSC) have the capacity to self-renew and differentiate in vitro into all the cell types of the organism, including the cardiac lineage. Thus, they provide a framework to study the early steps of human cardiac differentiation. The objective of the present work was to develop a reporter hPSC cell line for the induction of the cardiac mesoderm, a key and early step in the process of cardiac differentiation. For this, we selected MESP1, a crucial transcription factor expressed in these precursor cells. We devised a strategy to insert the green fluorescent protein mEmerald into the endogenous locus of MESP1 using CRISPR/Cas9 through homologous directed repair (HDR), generating a fusion protein connected to this transcription factor through a linker peptide. With this objective, we first designed two guide RNAs targeting the STOP codon within the second exon of MESP1 and cloned them into a vector that expresses the Cas9 protein. Next, we designed the HDR donor vector to contain the mEmerald sequence together with a floxed G418 resistance expression cassette, both flanked by two MESP1 800 bp homology arms. After successfully generating the HDR donor vector, we transfected it in hPSCS together with either plasmid gRNAs or both combined and selected three independent G418 resistant cell lines. Genomic PCR analysis of these lines with primers flanking the mEmerald/G418 cassette integration site indicated the successful homologous recombination in these cell lines. Since each of these lines consists of a pool of genotypically distinct cells, we next isolated 7 clonal cell lines for further examination. We are currently evaluating the correct integration of mEmerald into the clones through PCR, and we will next assess the expression of this fluorescent protein during cardiac differentiation protocols through microscopy and flow cytometry. Once validated, this reporter cell line will be a very valuable tool for assessing the early stages of cardiac induction