Fluorescent Gene Tagging of Transcriptionally Silent Genes in hiPSCs.

Authors :: Roberts B
Hendershott MC
Arakaki J
Gerbin KA
Malik H
Nelson A
Gehring J
Hookway C
Ludmann SA
Yang R
Haupt A
Grancharova T
Valencia V
Fuqua MA
Tucker A
Rafelski SM
Gunawardane RN
Source :: Stem cell reports [Stem Cell Reports] 2019 May 14; Vol. 12 (5), pp. 1145-1158. Date of Electronic Publication: 2019 Apr 04.
Publication Year :: 2019
Abstract: 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.<br /> (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)