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Efficient Gene Editing of Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

  • Saniye Yumlu
  • Sanum Bashir
  • Jürgen Stumm
  • Ralf KühnEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1961)

Abstract

The generation of targeted mutants is a crucial step toward studying the biomedical effect of genes of interest. The generation of such mutants in human induced pluripotent stem cells (iPSCs) is of an utmost importance as these cells carry the potential to be differentiated into any cell lineage. Using the CRISPR/Cas9 nuclease system for induction of targeted double-strand breaks, gene editing of target loci in iPSCs can be achieved with high efficiency. This chapter covers protocols for the preparation of reagents to target loci of interest, the transfection, and for the genotyping of single cell-derived iPSC clones. Furthermore, we provide a protocol for the convenient generation of plasmids enabling multiplex gene targeting.

Key words

Pluripotent stem cells Gene editing CRISPR Cas9 Knockout Knockin i53 Trex2 

References

  1. 1.
    Shui B, Hernandez Matias L, Guo Y, Peng Y (2016) The rise of CRISPR/Cas for genome editing in stem cells. Stem Cells Int 2016:8140168. https://doi.org/10.1155/2016/8140168CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Jinek M, Chylinski K, Fonfara I et al (2012) A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 337:816. LP-821CrossRefGoogle Scholar
  3. 3.
    Barrangou R (2014) Cas9 targeting and the CRISPR revolution. Science 344:707. LP-708CrossRefGoogle Scholar
  4. 4.
    Hess GT, Tycko J, Yao D, Bassik MC (2017) Methods and applications of CRISPR-mediated base editing in eukaryotic genomes. Mol Cell 68:26–43. https://doi.org/10.1016/j.molcel.2017.09.029CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157:1262–1278. https://doi.org/10.1016/j.cell.2014.05.010CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Ma D, Liu F (2015) Genome editing and its applications in model organisms. Genom Proteom Bioinformat 13:336–344. https://doi.org/10.1016/j.gpb.2015.12.001CrossRefGoogle Scholar
  7. 7.
    Chu VT, Weber T, Wefers B et al (2015) Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol 33:543–548. https://doi.org/10.1038/nbt.3198CrossRefPubMedGoogle Scholar
  8. 8.
    Kim EJ, Kang KH, Ju JH (2017) CRISPR-Cas9: a promising tool for gene editing on induced pluripotent stem cells. Korean J Intern Med 32:42–61. https://doi.org/10.3904/kjim.2016.198CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Yang D, Scavuzzo MA, Chmielowiec J et al (2016) Enrichment of G2/M cell cycle phase in human pluripotent stem cells enhances HDR-mediated gene repair with customizable endonucleases. Sci Rep 6:1–15. https://doi.org/10.1038/srep21264CrossRefGoogle Scholar
  10. 10.
    Li H, Beckman KA, Pessino V, et al (2017) Design and specificity of long ssDNA donors for CRISPR-based knock-in. https://doi.org/10.1101/178905
  11. 11.
    Yumlu S, Stumm J, Bashir S et al (2017) Gene editing and clonal isolation of human induced pluripotent stem cells using CRISPR/Cas9. Methods 121–122:29–44. https://doi.org/10.1016/j.ymeth.2017.05.009CrossRefPubMedGoogle Scholar
  12. 12.
    Mohr SE, Hu Y, Ewen-Campen B et al (2016) CRISPR guide RNA design for research applications. FEBS J 283:3232–3238. https://doi.org/10.1111/febs.13777CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Merkle FT, Neuhausser WM, Santos D et al (2015) Efficient CRISPR-Cas9-mediated generation of knockin human pluripotent stem cells lacking undesired mutations at the targeted locus. Cell Rep 11:875–883. https://doi.org/10.1016/j.celrep.2015.04.007CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Cui Y, Xu J, Cheng M et al (2018) Review of CRISPR/Cas9 sgRNA design tools. Interdiscip Sci 10(2):455–465. https://doi.org/10.1007/s12539-018-0298-zCrossRefPubMedGoogle Scholar
  15. 15.
    Peters D, Cowan C, Musunuru K (2014) Genome editing in human pluripotent stem cells. In: StemBook. Harvard Stem Cell Institute, Cambridge, MA. https://doi.org/10.3824/stembook.1.94.1CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Saniye Yumlu
    • 1
    • 2
  • Sanum Bashir
    • 1
    • 2
  • Jürgen Stumm
    • 1
    • 2
  • Ralf Kühn
    • 1
    • 2
    Email author
  1. 1.Max-Delbrück-Centrum für Molekulare MedizinBerlinGermany
  2. 2.Berlin Institute of HealthBerlinGermany

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