Generation of Reporter Human Pluripotent Stem Cells Using CRISPR/Cas9 Editing (original) (raw)
- 167 Accesses
Abstract
Differentiated cells derived from human pluripotent stem cells (hPSCs) have found wide applications in modeling cellular transplantation therapies, evaluating drug candidates, and advancing basic biology. The precision monitoring and selection of specific differentiated cell types can be achieved by knocking-in a cassette encoding a fluorescent protein at the end of various differentiation marker genes using genome-editing techniques. Although genome editing with homology-directed repair has been advanced to knock-in desired reporter genes into the human genome, the efficiency is still low due to the random insertion of donor vectors into the host genome. We established a method to use a “double-tk donor vector system” in which the expression units of the thymidine kinase of Herpes simplex virus (HSV-tk) are placed on both outer sides of homology arms. This system is useful for selecting knocked-in hPSCs compared to conventional donor vector systems with no or a single HSV-tk cassette. In this chapter, we describe this method for the generation of reporter hPSCs using CRISPR/Cas9 editing technology with this cellular suicide system based on double-tk donor vectors.
References
- Thomson JA et al (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147
Article CAS PubMed Google Scholar - Takahashi K et al (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872. https://doi.org/10.1016/j.cell.2007.11.019
Article CAS PubMed Google Scholar - Yu J et al (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917–1920. https://doi.org/10.1126/science.1151526
Article CAS PubMed Google Scholar - Deltcheva E et al (2011) CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471:602–607. https://doi.org/10.1038/nature09886
Article CAS PubMed PubMed Central Google Scholar - Mali P et al (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826. https://doi.org/10.1126/science.1232033
Article CAS PubMed PubMed Central Google Scholar - Hou Z et al (2013) Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc Natl Acad Sci USA 110:15644–15649. https://doi.org/10.1073/pnas.1313587110
Article CAS PubMed PubMed Central Google Scholar - Zhang JP et al (2017) Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage. Genome Biol 18:35. https://doi.org/10.1186/s13059-017-1164-8
Article CAS PubMed PubMed Central Google Scholar - Anzai T et al (2021) Generation of efficient Knock-in mouse and human pluripotent stem cells using CRISPR-Cas9. Methods Mol Biol 2320:247–259. https://doi.org/10.1007/978-1-0716-1484-6_22
Article CAS PubMed Google Scholar - Lin S, Staahl BT, Alla RK, Doudna JA (2014) Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. elife 3:e04766. https://doi.org/10.7554/eLife.04766
Article PubMed PubMed Central Google Scholar - Maurissen TL, Woltjen K (2020) Synergistic gene editing in human iPS cells via cell cycle and DNA repair modulation. Nat Commun 11:2876. https://doi.org/10.1038/s41467-020-16643-5
Article CAS PubMed PubMed Central Google Scholar - Arnoult N et al (2017) Regulation of DNA repair pathway choice in S and G2 phases by the NHEJ inhibitor CYREN. Nature 549:548–552. https://doi.org/10.1038/nature24023
Article CAS PubMed PubMed Central Google Scholar - Nambiar TS et al (2019) Stimulation of CRISPR-mediated homology-directed repair by an engineered RAD18 variant. Nat Commun 10:3395. https://doi.org/10.1038/s41467-019-11105-z
Article CAS PubMed PubMed Central Google Scholar - Haapaniemi E, Botla S, Persson J, Schmierer B, Taipale J (2018) CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response. Nat Med 24:927–930. https://doi.org/10.1038/s41591-018-0049-z
Article CAS PubMed Google Scholar - Ihry RJ et al (2018) p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells. Nat Med 24:939–946. https://doi.org/10.1038/s41591-018-0050-6
Article CAS PubMed Google Scholar - Hockemeyer D et al (2009) Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat Biotechnol 27:851–857. https://doi.org/10.1038/nbt.1562
Article CAS PubMed PubMed Central Google Scholar - Ashton WT, Karkas JD, Field AK, Tolman RL (1982) Activation by thymidine kinase and potent antiherpetic activity of 2′-nor-2′-deoxyguanosine (2’NDG). Biochem Biophys Res Commun 108:1716–1721. https://doi.org/10.1016/s0006-291x(82)80109-5
Article CAS PubMed Google Scholar - Smith KO, Galloway KS, Kennell WL, Ogilvie KK, Radatus BK (1982) A new nucleoside analog, 9-[[2-hydroxy-1-(hydroxymethyl)ethoxyl]methyl]guanine, highly active in vitro against herpes simplex virus types 1 and 2. Antimicrob Agents Chemother 22:55–61. https://doi.org/10.1128/AAC.22.1.55
Article CAS PubMed PubMed Central Google Scholar - Moolten FL (1986) Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes—paradigm for a prospective cancer control strategy. Cancer Res 46:5276–5281
CAS PubMed Google Scholar - Schwartz F et al (1991) A dominant positive and negative selectable gene for use in mammalian-cells. Proc Natl Acad Sci USA 88:10416–10420. https://doi.org/10.1073/pnas.88.23.10416
Article CAS PubMed PubMed Central Google Scholar - Naujok O, Kaldrack J, Taivankhuu T, Jorns A, Lenzen S (2010) Selective removal of undifferentiated embryonic stem cells from differentiation cultures through HSV1 thymidine kinase and ganciclovir treatment. Stem Cell Rev Rep 6:450–461. https://doi.org/10.1007/s12015-010-9148-z
Article CAS PubMed Google Scholar - Iwasawa C et al (2019) Increased cytotoxicity of herpes simplex virus thymidine kinase expression in human induced pluripotent stem cells. Int J Mol Sci 20:810
Article CAS PubMed PubMed Central Google Scholar - Nakade K et al (2023) Efficient selection of knocked-in pluripotent stem cells using dual cassettes of cellular suicide system. Available at SSRN: https://ssrn.com/abstract=4290057 or https://doi.org/10.2139/ssrn.4290057
- Tsukamoto S et al (2021) Generation of two ISL1-tdTomato reporter human induced pluripotent stem cell lines using CRISPR-Cas9 genome editing. Stem Cell Res 53:102363
Article CAS PubMed Google Scholar
Acknowledgments
We would like to express our sincere gratitude to Ms. Kumiko Omori for her administrative support. This research was supported in part by the grants from AMED (23bm1423010h0001) to YH and Uehara Memorial Foundation to YH.
Author information
Authors and Affiliations
- iPS Cell Advanced Characterization and Development Team, RIKEN BioResource Research Center, Ibaraki, Japan
Yohei Hayashi - Gene Engineering Division, RIKEN BioResource Research Center, Ibaraki, Japan
Koji Nakade
Authors
- Yohei Hayashi
You can also search for this author inPubMed Google Scholar - Koji Nakade
You can also search for this author inPubMed Google Scholar
Corresponding author
Correspondence toYohei Hayashi .
Editor information
Editors and Affiliations
- Department of Biochemistry & Molecular Biology, Louisiana State University Health Sciences, Shreveport, LA, USA
Baojin Ding - Department of Geriatrics - Xiangya Hospital, Central South University, Changsha City, Hunan, China
Yu Tang
Rights and permissions
Copyright information
© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Hayashi, Y., Nakade, K. (2024). Generation of Reporter Human Pluripotent Stem Cells Using CRISPR/Cas9 Editing. In: Ding, B., Tang, Y. (eds) Human Induced Pluripotent Stem Cells. Neuromethods, vol 210. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3999-3\_7
Download citation
- .RIS
- .ENW
- .BIB
- DOI: https://doi.org/10.1007/978-1-0716-3999-3\_7
- Published: 14 July 2024
- Publisher Name: Humana, New York, NY
- Print ISBN: 978-1-0716-3998-6
- Online ISBN: 978-1-0716-3999-3
- eBook Packages: Springer Protocols