PT  - JOURNAL ARTICLE
AU  - Brock Roberts
AU  - Joy Arakaki
AU  - Kaytlyn A. Gerbin
AU  - Haseeb Malik
AU  - Angelique Nelson
AU  - Melissa C. Hendershott
AU  - Caroline Hookway
AU  - Susan A. Ludmann
AU  - Irina A. Mueller
AU  - Ruian Yang
AU  - Susanne M. Rafelski
AU  - Ruwanthi N. Gunawardane
TI  - Scarless gene tagging of transcriptionally silent genes in hiPSCs to visualize cardiomyocyte sarcomeres in live cells
AID  - 10.1101/342881
DP  - 2018 Jan 01
TA  - bioRxiv
PG  - 342881
4099  - http://biorxiv.org/content/early/2018/06/09/342881.short
4100  - http://biorxiv.org/content/early/2018/06/09/342881.full
AB  - 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.