Fluorescent Gene Tagging of Transcriptionally Silent Genes in HiPSCs

Overview

Journal Stem Cell Reports

Publisher Cell Press

Specialty Cell Biology

Date 2019 Apr 9

PMID 30956114

Citations 25

Authors

Brock Roberts

Melissa C Hendershott

Joy Arakaki

Kaytlyn A Gerbin

Haseeb Malik

Angelique Nelson

Jamie Gehring

Caroline Hookway

Susan A Ludmann

Ruian Yang

Amanda Haupt

Tanya Grancharova

Veronica Valencia

Margaret A Fuqua

Andrew Tucker

Susanne M Rafelski

Ruwanthi N Gunawardane

Affiliations

Soon will be listed here.

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.

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References

Doyon J, Zeitler B, Cheng J, Cheng A, Cherone J, Santiago Y . Rapid and efficient clathrin-mediated endocytosis revealed in genome-edited mammalian cells. Nat Cell Biol. 2011; 13(3):331-7. PMC: 4113319. DOI: 10.1038/ncb2175. View

Luther P . The vertebrate muscle Z-disc: sarcomere anchor for structure and signalling. J Muscle Res Cell Motil. 2009; 30(5-6):171-85. PMC: 2799012. DOI: 10.1007/s10974-009-9189-6. View

Palpant N, Pabon L, Roberts M, Hadland B, Jones D, Jones C . Inhibition of β-catenin signaling respecifies anterior-like endothelium into beating human cardiomyocytes. Development. 2015; 142(18):3198-209. PMC: 4582173. DOI: 10.1242/dev.117010. View

Kim S, Matsumoto T, Kagawa H, Nakamura M, Hirohata R, Ueno A . Microhomology-assisted scarless genome editing in human iPSCs. Nat Commun. 2018; 9(1):939. PMC: 5838097. DOI: 10.1038/s41467-018-03044-y. View

Lackner D, Carre A, Guzzardo P, Banning C, Mangena R, Henley T . A generic strategy for CRISPR-Cas9-mediated gene tagging. Nat Commun. 2015; 6:10237. PMC: 4703899. DOI: 10.1038/ncomms10237. View

Roberts B, Haupt A, Tucker A, Grancharova T, Arakaki J, Fuqua M . Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization. Mol Biol Cell. 2017; 28(21):2854-2874. PMC: 5638588. DOI: 10.1091/mbc.E17-03-0209. View

Otsuka S, Bui K, Schorb M, Hossain M, Politi A, Koch B . Nuclear pore assembly proceeds by an inside-out extrusion of the nuclear envelope. Elife. 2016; 5. PMC: 5065316. DOI: 10.7554/eLife.19071. View

Kreitzer F, Salomonis N, Sheehan A, Huang M, Park J, Spindler M . A robust method to derive functional neural crest cells from human pluripotent stem cells. Am J Stem Cells. 2013; 2(2):119-31. PMC: 3708511. View

Palpant N, Hofsteen P, Pabon L, Reinecke H, Murry C . Cardiac development in zebrafish and human embryonic stem cells is inhibited by exposure to tobacco cigarettes and e-cigarettes. PLoS One. 2015; 10(5):e0126259. PMC: 4433280. DOI: 10.1371/journal.pone.0126259. View

10.

Grassart A, Cheng A, Hong S, Zhang F, Zenzer N, Feng Y . Actin and dynamin2 dynamics and interplay during clathrin-mediated endocytosis. J Cell Biol. 2014; 205(5):721-35. PMC: 4050722. DOI: 10.1083/jcb.201403041. View

11.

Lian X, Bao X, Zilberter M, Westman M, Fisahn A, Hsiao C . Chemically defined, albumin-free human cardiomyocyte generation. Nat Methods. 2015; 12(7):595-6. PMC: 4663075. DOI: 10.1038/nmeth.3448. View

12.

Dambournet D, Hong S, Grassart A, Drubin D . Tagging endogenous loci for live-cell fluorescence imaging and molecule counting using ZFNs, TALENs, and Cas9. Methods Enzymol. 2014; 546:139-60. DOI: 10.1016/B978-0-12-801185-0.00007-6. View

13.

Haupt A, Grancharova T, Arakaki J, Fuqua M, Roberts B, Gunawardane R . Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9. J Vis Exp. 2018; (138). PMC: 6231893. DOI: 10.3791/58130. View

14.

Gerbin K, Yang X, Murry C, Coulombe K . Enhanced Electrical Integration of Engineered Human Myocardium via Intramyocardial versus Epicardial Delivery in Infarcted Rat Hearts. PLoS One. 2015; 10(7):e0131446. PMC: 4498815. DOI: 10.1371/journal.pone.0131446. View

15.

Yao Z, Mich J, Ku S, Menon V, Krostag A, Martinez R . A Single-Cell Roadmap of Lineage Bifurcation in Human ESC Models of Embryonic Brain Development. Cell Stem Cell. 2017; 20(1):120-134. PMC: 5261831. DOI: 10.1016/j.stem.2016.09.011. View

16.

Gibson T, Seiler M, Veitia R . The transience of transient overexpression. Nat Methods. 2013; 10(8):715-21. DOI: 10.1038/nmeth.2534. View

17.

Skarnes W, Rosen B, West A, Koutsourakis M, Bushell W, Iyer V . A conditional knockout resource for the genome-wide study of mouse gene function. Nature. 2011; 474(7351):337-42. PMC: 3572410. DOI: 10.1038/nature10163. View

18.

Shen M, Arbab M, Hsu J, Worstell D, Culbertson S, Krabbe O . Predictable and precise template-free CRISPR editing of pathogenic variants. Nature. 2018; 563(7733):646-651. PMC: 6517069. DOI: 10.1038/s41586-018-0686-x. View

19.

Nakade S, Tsubota T, Sakane Y, Kume S, Sakamoto N, Obara M . Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9. Nat Commun. 2014; 5:5560. PMC: 4263139. DOI: 10.1038/ncomms6560. View

20.

Sakuma T, Nakade S, Sakane Y, Suzuki K, Yamamoto T . MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems. Nat Protoc. 2015; 11(1):118-33. DOI: 10.1038/nprot.2015.140. View