Abstract
We describe a multi-step CRISPR/Cas9 gene editing method to create endogenously tagged GFP-fusions of transcriptionally silent genes in human induced pluripotent stem cells (hiPSCs), allowing visualization of proteins that are only expressed upon differentiation. To do this, we designed a donor template containing the monomeric EGFP (mEGFP) fusion tag and an mCherry selection cassette delivered in tandem to a target locus via homology directed repair (HDR). mCherry expression was driven by a constitutive promoter and served as a drug-free, excisable selection marker. Following selection, the mCherry cassette was excised with Cas9, creating an mEGFP-fusion with the target gene. We achieved scarless excision by using repetitive sequences to guide microhomology-mediated end joining (MMEJ) and introduce linker sequences between the mEGFP tag and the target gene. Using this strategy, we successfully tagged genes encoding the cardiomyocyte sarcomeric proteins troponin I (TNNI1), alpha-actinin (ACTN2), titin (TTN), myosin light chain 2a (MYL7), and myosin light chain 2v (MYL2) with mEGFP in undifferentiated hiPSCs. This methodology provides a general strategy for scarlessly introducing tags to transcriptionally silent loci in hiPSCs.
