Advertisement

CRISPR/Cas-Mediated Knockin in Human Pluripotent Stem Cells

  • Nipun Verma
  • Zengrong Zhu
  • Danwei HuangfuEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1513)

Abstract

Fluorescent reporter and epitope-tagged human pluripotent stem cells (hPSCs) greatly facilitate studies on the pluripotency and differentiation characteristics of these cells. Unfortunately traditional procedures to generate such lines are hampered by a low targeting efficiency that necessitates a lengthy process of selection followed by the removal of the selection cassette. Here we describe a procedure to generate fluorescent reporter and epitope tagged hPSCs in an efficient one-step process using the CRISPR/Cas technology. Although the method described uses our recently developed iCRISPR platform, the protocols can be adapted for general use with CRISPR/Cas or other engineered nucleases. The transfection procedures described could also be used for additional applications, such as overexpression or lineage tracing studies.

Key words

Human pluripotent stem cells (hPSCs) Gene targeting CRISPR/Cas Homologous recombination Knockin Fluorescent reporter Epitope tag 

Notes

Acknowledgements

Nipun Verma and Zengrong Zhu contributed equally to this work. Our work related to this publication was funded, in part, by NIH (R01DK096239) and NYSTEM (C029156). Z.Z. was supported by the New York State Stem Cell Science (NYSTEM) fellowship from the Center for Stem Cell Biology (CSCB) of the Sloan Kettering Institute. N.V. was supported by the Howard Hughes Medical Institute (HHMI) Medical Research and the Tri-Institutional Weill Cornell/ Rockefeller/ Sloan Kettering MD-PhD program.

References

  1. 1.
    Tabar V, Studer L (2014) Pluripotent stem cells in regenerative medicine: challenges and recent progress. Nat Rev Genet 15:82–92CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Zhu Z, Huangfu D (2013) Human pluripotent stem cells: an emerging model in developmental biology. Development 140:705–717CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Chia NY, Chan YS, Feng B et al (2010) A genome-wide RNAi screen reveals determinants of human embryonic stem cell identity. Nature 468:316–320CrossRefPubMedGoogle Scholar
  4. 4.
    Theunissen TW, Powell BE, Wang H et al (2014) Systematic identification of culture conditions for induction and maintenance of naive human pluripotency. Cell Stem Cell 15:471–487CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Eiges R, Schuldiner M, Drukker M et al (2001) Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells. Curr Biol 11:514–518CrossRefPubMedGoogle Scholar
  6. 6.
    Zwaka TP, Thomson JA (2003) Homologous recombination in human embryonic stem cells. Nat Biotechnol 21:319–321CrossRefPubMedGoogle Scholar
  7. 7.
    Davis RP, Costa M, Grandela C et al (2008) A protocol for removal of antibiotic resistance cassettes from human embryonic stem cells genetically modified by homologous recombination or transgenesis. Nat Protoc 3:1550–1558CrossRefPubMedGoogle Scholar
  8. 8.
    Zhu Z, Verma N, Gonzalez F, Shi ZD, Huangfu D (2015) A CRISPR/Cas-mediated selection-free knockin strategy in human embryonic stem cells. Stem Cell Rep 4:1103–1111CrossRefGoogle Scholar
  9. 9.
    Joung JK, Sander JD (2013) TALENs: a widely applicable technology for targeted genome editing. Nat Rev Mol Cell Biol 14:49–55CrossRefPubMedGoogle Scholar
  10. 10.
    Ran FA, Hsu PD, Wright J et al (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8:2281–2308CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Urnov FD, Rebar EJ, Holmes MC et al (2010) Genome editing with engineered zinc finger nucleases. Nat Rev Genet 11:636–646CrossRefPubMedGoogle Scholar
  12. 12.
    Jinek M, Chylinski K, Fonfara I et al (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821CrossRefPubMedGoogle Scholar
  13. 13.
    Jinek M, East A, Cheng A et al (2013) RNA-programmed genome editing in human cells. Elife 2:e00471CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Cho SW, Kim S, Kim JM, Kim JS (2013) Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 31:230–232CrossRefPubMedGoogle Scholar
  15. 15.
    Cong L, Ran FA, Cox D et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Mali P, Yang L, Esvelt KM et al (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Byrne SM, Ortiz L, Mali P et al (2015) Multi-kilobase homozygous targeted gene replacement in human induced pluripotent stem cells. Nucleic Acids Res 43:e21CrossRefPubMedGoogle Scholar
  18. 18.
    Hou Z, Zhang Y, Propson NE et al (2013) Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc Natl Acad Sci U S A 110:15644–15649CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Merkert S, Wunderlich S, Bednarski C et al (2014) Efficient designer nuclease-based homologous recombination enables direct PCR screening for footprintless targeted human pluripotent stem cells. Stem Cell Rep 2:107–118CrossRefGoogle Scholar
  20. 20.
    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 11:875–883CrossRefPubMedGoogle Scholar
  21. 21.
    Gonzalez F, Zhu Z, Shi ZD et al (2014) An iCRISPR platform for rapid, multiplexable, and inducible genome editing in human pluripotent stem cells. Cell Stem Cell 15:215–226CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Zhu Z, Gonzalez F, Huangfu D (2014) The iCRISPR platform for rapid genome editing in human pluripotent stem cells. Methods Enzymol 546:215–250CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Chen G, Gulbranson DR, Hou Z et al (2011) Chemically defined conditions for human iPSC derivation and culture. Nat Methods 8:424–429CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Ludwig TE, Bergendahl V, Levenstein ME et al (2006) Feeder-independent culture of human embryonic stem cells. Nat Methods 3:637–646CrossRefPubMedGoogle Scholar
  25. 25.
    Veres A, Gosis BS, Ding Q et al (2014) Low incidence of off-target mutations in individual CRISPR-Cas9 and TALEN targeted human stem cell clones detected by whole-genome sequencing. Cell Stem Cell 15:27–30CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Borjigin J, Nathans J (1994) Insertional mutagenesis as a probe of rhodopsin's topography, stability, and activity. J Biol Chem 269:14715–14722PubMedGoogle Scholar
  27. 27.
    Grote E, Hao JC, Bennett MK, Kelly RB (1995) A targeting signal in VAMP regulating transport to synaptic vesicles. Cell 81:581–589CrossRefPubMedGoogle Scholar
  28. 28.
    Nieminen M, Tuuri T, Savilahti H (2010) Genetic recombination pathways and their application for genome modification of human embryonic stem cells. Exp Cell Res 316:2578–2586CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Developmental Biology ProgramSloan Kettering InstituteNew YorkUSA
  2. 2.Weill Graduate School of Medical Sciences at Cornell University/The Rockefeller University/Sloan Kettering Institute Tri-Institutional M.D.-Ph.D. ProgramNew YorkUSA

Personalised recommendations