Fluorescent Gene Tagging of Transcriptionally Silent Genes in hiPSCs

Abstract

Summary We describe a multistep method for endogenous tagging of transcriptionally silent genes in human induced pluripotent stem cells (hiPSCs). A monomeric EGFP (mEGFP) fusion tag and a constitutively expressed mCherry fluorescence selection cassette were delivered in tandem via homology-directed repair to five genes not expressed in hiPSCs but important for cardiomyocyte sarcomere function: TTN , MYL7 , MYL2 , TNNI1 , and ACTN2 . CRISPR/Cas9 was used to deliver the selection cassette and subsequently mediate its excision via microhomology-mediated end-joining and non-homologous end-joining. Most excised clones were effectively tagged, and all properly tagged clones expressed the mEGFP fusion protein upon differentiation into cardiomyocytes, allowing live visualization of these cardiac proteins at the sarcomere. This methodology provides a broadly applicable strategy for endogenously tagging transcriptionally silent genes in hiPSCs, potentially enabling their systematic and dynamic study during differentiation and morphogenesis.

Cell states beyond transcriptomics: integrating structural organization and gene expression in hiPSC-derived cardiomyocytes

“…This offered an opportunity to test the proposition that RNA abundance levels correlate with cell organization (Battich et al, 2015;Gut et al, 2018;Popovic et al, 2018). We used an hiPSC line expressing mEGFP-tagged alpha-actinin-2 (ACTN2-mEGFP; Roberts et al, 2019), which localizes to the z-disk of the sarcomere, to quantify and stage sarcomere organization (Luther et al, 2011). The differentiated cardiomyocyte populations were replated for high resolution imaging and revealed a broad range of sarcomere organization with a general increase in myofibril organization over time ( Figure 3A, B).…”

Profiling parvalbumin interneurons using iPSC: challenges and perspectives for Autism Spectrum Disorder (ASD)

“…Generation of tagged-parvalbumin neurons by a CRISPR/Cas9 approach as a tool to follow PVALB neuron cell fate The CRISPR/Cas9 genome-editing technology (reviewed in [53]) has been broadly used to modify iPSC and recently, many studies successfully demonstrated the generation of human iPSC lines expressing a fluorescent protein that faithfully recapitulates the endogenous expression of a given protein [54,55]. Through a CRISPR/Cas9 approach, one could insert a reporter cassette (i.e., eGFP, tdTomato), whose expression is driven by the endogenous PVALB promoter, allowing to follow the fate of these neurons once PV expression starts.…”

Probing pluripotency gene regulatory networks with quantitative live cell imaging

“…Incomplete characterization of cell lines and insufficient reporting of details for creating and validating cell lines is common and may be an impediment to their adoption by others. While cell line characterization is quite extensive in some reports [14] , [17] , [26] , [96] too few are sufficiently documented. This failure leads to a lack of confidence in the biological significance of the data generated using these cell lines.…”

“…This failure leads to a lack of confidence in the biological significance of the data generated using these cell lines. Exemplars of thorough cell line validation and characterization include the Allen Institute for Cell Science (AICS) [96] and the European Molecular Biology Laboratory (EMBL) [87] . Both groups focus on the complete description of the reporter construction, the use of multiple methodologies to interrogate the details of the genomic editing accuracy and thorough evaluation of the functional properties of the reporter that relate to its ability to act as a biologically relevant expression surrogate for the GOI.…”