Scarless gene tagging of transcriptionally silent genes in hiPSCs to visualize cardiomyocyte sarcomeres in live cells

Roberts, B.; Arakaki, J.; Gerbin, K. A.; Malik, H.; Nelson, A.; Hendershott, M. C.; Hookway, C.; Ludmann, S. A.; Mueller, I. A.; Yang, R.; Rafelski, S. M.; Gunawardane, R. N.

doi:10.1101/342881

bioRxiv · 10.1101/342881

Scarless gene tagging of transcriptionally silent genes in hiPSCs to visualize cardiomyocyte sarcomeres in live cells

Roberts, B.·Arakaki, J.·Gerbin, K. A.·Malik, H.·Nelson, A.·Hendershott, M. C.·Hookway, C.·Ludmann, S. A.·Mueller, I. A.·Yang, R.·Rafelski, S. M.·Gunawardane, R. N.

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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.

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Roberts, B., Arakaki, J., Gerbin, K. A., Malik, H., Nelson, A., Hendershott, M. C., Hookway, C., Ludmann, S. A., Mueller, I. A., Yang, R., Rafelski, S. M., Gunawardane, R. N.. 2018-06-09. Scarless gene tagging of transcriptionally silent genes in hiPSCs to visualize cardiomyocyte sarcomeres in live cells. https://doi.org/10.1101/342881

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