Skip to main content
SpringerLink
Log in

Reprogramming of Fibroblasts to Human iPSCs by CRISPR Activators

  • Protocol
  • First Online:
Nuclear Reprogramming

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2239))

Abstract

CRISPR-mediated gene activation (CRISPRa) can be used to target endogenous genes for activation. By targeting pluripotency-associated reprogramming factors, human fibroblasts can be reprogrammed into induced pluripotent stem cells (iPSCs). Here, we describe a method for the derivation of iPSCs from human fibroblasts using episomal plasmids encoding CRISPRa components. This chapter also provides procedure to assemble guide RNA cassettes and generation of multiplexed guide plasmids for readers who want to design their own guide RNAs.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126(4):663–676. https://doi.org/10.1016/j.cell.2006.07.024

    Article  CAS  PubMed  Google Scholar 

  2. Anokye-Danso F, Trivedi CM, Juhr D, Gupta M, Cui Z, Tian Y, Zhang YZ, Yang WL, Gruber PJ, Epstein JA, Morrisey EE (2011) Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Cell Stem Cell 8(4):376–388. https://doi.org/10.1016/j.stem.2011.03.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Miyoshi N, Ishii H, Nagano H, Haraguchi N, Dewi DL, Kano Y, Nishikawa S, Tanemura M, Mimori K, Tanaka F, Saito T, Nishimura J, Takemasa I, Mizushima T, Ikeda M, Yamamoto H, Sekimoto M, Doki Y, Mori M (2011) Reprogramming of mouse and human cells to pluripotency using mature microRNAs. Cell Stem Cell 8(6):633–638. https://doi.org/10.1016/j.stem.2011.05.001

    Article  CAS  PubMed  Google Scholar 

  4. Hou P, Li Y, Zhang X, Liu C, Guan J, Li H, Zhao T, Ye J, Yang W, Liu K, Ge J, Xu J, Zhang Q, Zhao Y, Deng H (2013) Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds. Science 341(6146):651–654. https://doi.org/10.1126/science.1239278

    Article  CAS  PubMed  Google Scholar 

  5. Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, Stern-Ginossar N, Brandman O, Whitehead EH, Doudna JA, Lim WA, Weissman JS, Qi LS (2013) CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154(2):442–451. https://doi.org/10.1016/j.cell.2013.06.044

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Maeder ML, Linder SJ, Cascio VM, Fu Y, Ho QH, Joung JK (2013) CRISPR RNA-guided activation of endogenous human genes. Nat Methods 10(10):977–979. https://doi.org/10.1038/nmeth.2598

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Perez-Pinera P, Kocak DD, Vockley CM, Adler AF, Kabadi AM, Polstein LR, Thakore PI, Glass KA, Ousterout DG, Leong KW, Guilak F, Crawford GE, Reddy TE, Gersbach CA (2013) RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat Methods 10(10):973–976. https://doi.org/10.1038/nmeth.2600

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Cheng AW, Wang H, Yang H, Shi L, Katz Y, Theunissen TW, Rangarajan S, Shivalila CS, Dadon DB, Jaenisch R (2013) Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res 23(10):1163–1171. https://doi.org/10.1038/cr.2013.122

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Liu P, Chen M, Liu Y, Qi LS, Ding S (2018) CRISPR-based chromatin remodeling of the endogenous Oct4 or Sox2 locus enables reprogramming to pluripotency. Cell Stem Cell 22(2):252–261, e254. https://doi.org/10.1016/j.stem.2017.12.001

    Article  CAS  PubMed  Google Scholar 

  10. Weltner J, Balboa D, Katayama S, Bespalov M, Krjutskov K, Jouhilahti EM, Trokovic R, Kere J, Otonkoski T (2018) Human pluripotent reprogramming with CRISPR activators. Nat Commun 9(1):2643. https://doi.org/10.1038/s41467-018-05067-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5):861–872. https://doi.org/10.1016/j.cell.2007.11.019

    Article  CAS  PubMed  Google Scholar 

  12. Park IH, Zhao R, West JA, Yabuuchi A, Huo H, Ince TA, Lerou PH, Lensch MW, Daley GQ (2008) Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451(7175):141–146. https://doi.org/10.1038/nature06534

    Article  CAS  PubMed  Google Scholar 

  13. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318(5858):1917–1920. https://doi.org/10.1126/science.1151526

    Article  CAS  PubMed  Google Scholar 

  14. Fusaki N, Ban H, Nishiyama A, Saeki K, Hasegawa M (2009) Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc Jpn Acad B Phys Biol Sci 85(8):348–362. https://doi.org/10.2183/pjab.85.348

    Article  CAS  Google Scholar 

  15. Yu J, Hu K, Smuga-Otto K, Tian S, Stewart R, Slukvin II, Thomson JA (2009) Human induced pluripotent stem cells free of vector and transgene sequences. Science 324(5928):797–801. https://doi.org/10.1126/science.1172482

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Warren L, Manos PD, Ahfeldt T, Loh YH, Li H, Lau F, Ebina W, Mandal PK, Smith ZD, Meissner A, Daley GQ, Brack AS, Collins JJ, Cowan C, Schlaeger TM, Rossi DJ (2010) Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7(5):618–630. https://doi.org/10.1016/j.stem.2010.08.012

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Okita K, Matsumura Y, Sato Y, Okada A, Morizane A, Okamoto S, Hong H, Nakagawa M, Tanabe K, Tezuka K, Shibata T, Kunisada T, Takahashi M, Takahashi J, Saji H, Yamanaka S (2011) A more efficient method to generate integration-free human iPS cells. Nat Methods 8(5):409–412. https://doi.org/10.1038/nmeth.1591

    Article  CAS  PubMed  Google Scholar 

  18. Balboa D, Weltner J, Eurola S, Trokovic R, Wartiovaara K, Otonkoski T (2015) Conditionally stabilized dCas9 activator for controlling gene expression in human cell reprogramming and differentiation. Stem Cell Rep 5(3):448–459. https://doi.org/10.1016/j.stemcr.2015.08.001

    Article  CAS  Google Scholar 

  19. Tohonen V, Katayama S, Vesterlund L, Jouhilahti EM, Sheikhi M, Madissoon E, Filippini-Cattaneo G, Jaconi M, Johnsson A, Burglin TR, Linnarsson S, Hovatta O, Kere J (2015) Novel PRD-like homeodomain transcription factors and retrotransposon elements in early human development. Nat Commun 6:8207. https://doi.org/10.1038/ncomms9207

    Article  PubMed  PubMed Central  Google Scholar 

  20. Engler C, Kandzia R, Marillonnet S (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS One 3(11):e3647. https://doi.org/10.1371/journal.pone.0003647

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

We thank Biocenter Finland Biomedicum Stem Cell Center core facility for technical support. This work has been supported by the Jane and Aatos Erkko Foundation, Sigrid Jusélius Foundation, and the Academy of Finland.

Author information

Author notes

  1. Jere Weltner

    Present address: Department of Clinical Science, Intervention and Technology, Karolinska Institutet, Stockholm, Sweden

  2. Jere Weltner

    Present address: Division of Obstetrics and Gynecology, Karolinska Universitetssjukhuset, Stockholm, Sweden

Authors and Affiliations

  1. Stem Cells and Metabolism Research Program, Biomedicum Stem Cell Center, University of Helsinki, Helsinki, Finland

    Jere Weltner & Ras Trokovic

  2. Department of Clinical Science, Intervention and Technology, Karolinska Institutet, Stockholm, Sweden

    Jere Weltner

  3. Division of Obstetrics and Gynecology, Karolinska Universitetssjukhuset, Stockholm, Sweden

    Jere Weltner

Authors
  1. Jere Weltner
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ras Trokovic
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Jere Weltner or Ras Trokovic .

Editor information

Editors and Affiliations

  1. Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, USA

    Kejin Hu

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Weltner, J., Trokovic, R. (2021). Reprogramming of Fibroblasts to Human iPSCs by CRISPR Activators. In: Hu, K. (eds) Nuclear Reprogramming. Methods in Molecular Biology, vol 2239. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1084-8_12

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1084-8_12

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1083-1

  • Online ISBN: 978-1-0716-1084-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation