1. Fluorescent Gene Tagging of Transcriptionally Silent Genes in hiPSCs
- Author
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Veronica Valencia, Tanya Grancharova, Kaytlyn A. Gerbin, Angelique M. Nelson, Margaret A. Fuqua, Melissa C. Hendershott, Amanda Haupt, Ruian Yang, Jamie L. Gehring, Joy Arakaki, Haseeb Malik, Susanne M. Rafelski, Andrew Tucker, Susan A. Ludmann, Caroline Hookway, Brock Roberts, and Ruwanthi N. Gunawardane
- Subjects
Sarcomeres ,Resource ,0301 basic medicine ,DNA End-Joining Repair ,Green Fluorescent Proteins ,Induced Pluripotent Stem Cells ,Biology ,Biochemistry ,Cell Line ,Green fluorescent protein ,03 medical and health sciences ,0302 clinical medicine ,Genome editing ,Genetics ,Humans ,CRISPR ,Actinin ,Myocytes, Cardiac ,Amino Acid Sequence ,Induced pluripotent stem cell ,lcsh:QH301-705.5 ,Gene ,Gene Editing ,lcsh:R5-920 ,Sequence Homology, Amino Acid ,Cas9 ,Troponin I ,Cell Differentiation ,Cell Biology ,Fusion protein ,Cell biology ,030104 developmental biology ,Gene Expression Regulation ,lcsh:Biology (General) ,CRISPR-Cas Systems ,lcsh:Medicine (General) ,mCherry ,030217 neurology & neurosurgery ,Developmental Biology - 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. : Gunawardane and colleagues use CRISPR/Cas9 to deliver an excisable cassette to transcriptionally silent loci in hiPSCs, then accomplish excision of the cassette in a second step utilizing Cas9/CRISPR and the MMEJ and NHEJ DNA-repair pathways. Excision results in mEGFP tagging of the targeted loci. Upon differentiation, each of five tagged cell lines appropriately expresses a unique fluorescent fusion protein localized to the sarcomere in live cardiomyocytes. Keywords: CRISPR/Cas9, genome editing, cardiomyocyte differentiation, stem cells, iPSCs, MMEJ, live imaging, endogenous fluorescent tagging, mEGFP, HDR
- Published
- 2019