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  • br Materials and methods br Acknowledgements This


    Materials and methods
    Acknowledgements This work was supported by grants from Instituto de Salud Carlos III (PI 15/000227 to MC, Plataforma en Red de Recursos Biomoleculares y BioinformaticosPRB2 PT13/0001/0042) cofunded by FEDER.
    Resource table. Resource details Loss-of-function mutations in the PITX2 transcription factor gene have been shown to cause genetically inherited AF (Qiu et al., 2014; Wang et al., 2014; Zhou et al., 2013). PITX2 is specifically expressed in the left human atrium and hence, PITX2-linked AF may be based on misregulated gene expression in this heart chamber (Kirchhof et al., 2011). Human iPS cells can now selectively be differentiated into atrial cardiomyocytes implying that PITX2-regulated genes may be identified in this system, with implications for drug target discovery (Devalla et al., 2015). Direct reprogramming using a self-replicating RNA vector was used to generate an integration-free wild-type hiPSC line termed F1 (Fig. 1A; Yoshioka et al. (2013)). F1 cells displayed typical hiPSC morphology (Fig. 1B), a normal male karyotype (Fig. 1C), and robust expression of OCT4 and NANOG at the protein level (Fig. 1D). Transgene expression was undetectable in established F1 cells and hence, this was due to the endogenous activation of pluripotency genes to normal levels (Fig. 1E and not shown). F1 cells were capable of spontaneously differentiating into derivatives of the three germ layers as well as into cardiomyocytes (CMs) following directed cardiac induction (Fig. 1F). On this validated wild-type hiPSC background, CRISPR/Cas9-based targeted mutagenesis was employed to disrupt PITX2 in an isoform-independent manner (Fig. 1G, top). Genomic as well as RT-PCR and sequencing served to demonstrate the successful introduction of frame shift-causing deletion mutations on both cftr channel in two independent hiPSC clones, designated PITX2fs/fs-1 and -2 (Fig. 1G, bottom). Stimulation with retinoic acid (RA) may be used to redirect cardiac differentiation of hiPSCs from a default ventricular into an atrial fate (Devalla et al., 2015). Indeed, based on this rationale, data in Fig. S2A–C demonstrates that all cell lines under investigation could selectively be differentiated in a cardiac subtype-specific manner, i.e. form atrial or ventricular CMs at high efficiency depending on the presence or absence of RA during differentiation. A preliminary transcriptome analysis of these samples shows that atrial and ventricular hiPSC-CMs differ profoundly, with known chamber-specific genes marking the two populations (Fig. S2D, left and middle panels). Finally, plotting atrial WT versus merged PITX2fs/fs samples suggests significant differential gene expression in the knockout model, perhaps bearing potential for identifying novel AF drug targets with this approach (Fig. S2D, right). We currently do not know the percentage of specifically left atrial CMs in these preparations, or whether complete loss of PITX2 function would per se interfere with left atrial specification.
    Materials and methods
    Resource table. Resource details We have generated a double knockout Bag1 ES cell line by targeting the exon 2 of Bag1 gene using the CRISPR/Cas9 gene editing system (Fig. 1A). Mouse ES cells were transfected with pSpCas9(BB)-2A-GFP plasmids which contained sgRNA targeting the exon 2 of Bag1. The transfected cells were cultured for two days and then the presence of GFP expressing cells were sorted and selected by fluorescence flow cytometry. The sorted GFP+ cells were reseeded onto 0.1% gelatin-coated culture dishes to obtain single cell derived ES cell clones. Fifty-two individual ES cell clones were picked, isolated and expanded for further analysis by DNA sequencing of targeted regions. Twenty homozygous mutant ES cell clones were obtained and a bi-allelic 34-nucleotide-deleted and 38-nucleotide-deleted clone, Bag1KO-mESC (Fig. 1B,C), were chosen for further characterization.