ALTERNATIVE: QUAKING KNOCK OUT INDUCES AN ALTERED PLURIPOTENT STATE WITHOUT COMPROMISING CARDIAC MESODERM COMMITMENT

CAROLINA COLLI; GUADALUPE AMIN; SEVLEVER FEDERICO; MARIA AGUSTINA SCARAFÍA; ALAN MIQUEAS MÖBBS; LUCÍA NATALIA MORO; ARIEL WAISMAN; ALEJANDRO DAMIÁN LA GRECA; SANTIAGO GABRIEL MIRIUKA

Congreso; REUNIÓN ANUAL DE SOCIEDADES DE BIOCIENCIA 2022; 2022

Circular RNAs (circRNA) were found to participate in the differentiationof human pluripotent stem cells (PSC). They originate frombackspliced junctions during processing of pre-mRNAs, producingcovalently-closed stable molecules. Formation of circRNA dependson several factors, including the activity of the RNA binding proteinQuaking (QKI). QKI5 increases during epithelial-to-mesenchymaltransition (EMT), and therefore it could be necessary for the firststages of cardiac differentiation. To test this, we aimed to generateand characterize QKI knock out (KO) lines in PSC. For generatingthe KO cells (QKI-KO) we targeted a genomic region comprising thepromoter region and first exon of QKI gene using CRISPR, affectingexpression of relevant isoforms (5/6/7). We corroborated the successof our strategy by PCR and Sanger sequencing on genomicDNA and by RTqPCR and Western blot to confirm the absence ofQKI expression. Next, we studied the pluripotent state of QKI-KOcells by assessing their proliferation and differentiation capacities.We observed that, while wild type (WT) and KO cell lines were morphologicallyindistinguishable, QKI-KO cells showed an increasein cells in S phase compared to WT (51,3%±1.9 vs 41,1%±1.4),demonstrated by measuring EdU incorporation by flow cytometry.Differentiation to cardiac mesoderm was apparently not affected inQKI-KO cells as evaluated by expression analysis of EMT markers(EOMES, MIXL1, PDGFRα) 24, 48h and 72h after addition of CHIR.However, pluripotency marker genes (OCT4, SOX2 and NANOG)were not downregulated after CHIR addition, indicating that QKI-KOcells might have an altered exit from pluripotency. In summary, wesuccessfully generated a KO cell line for QKI that evinces increasedproliferation rates, sustained expression of pluripotent markersduring early EMT and differentiates into cardiomyocytes. Further experimentswill be directed to characterizing the different cardiac celltypes obtained compared to WT.