Skip to main content

In Vivo Validation of CRISPR Reagents in Preimplantation Mouse Embryos

Part of the Methods in Molecular Biology book series (MIMB,volume 2066)

Abstract

The CRISPR/Cas9 system has enjoyed enormous success and has now become the standard method of generating gene-modified mouse models. The tools for predicting the activity of CRISPR reagents in the mouse embryo are currently limited and not particularly accurate in predicting if a given reagent will be active. Given the time and cost of generating genetically modified mice, it is highly desirable to use CRISPR reagents that are known to be active in the mouse embryo. In this chapter, we provide a detailed procedure for empirically testing the activity of CRISPR reagents via electroporation into cultured preimplantation mouse embryos. This platform has proven to be rapid, efficient, and applicable to a variety of mouse strains, and can be used for assessing on- and off-target activity through a variety of molecular assays.

Key words

  • CRISPR
  • Validation
  • crRNA
  • gRNA
  • Indels
  • Cas9
  • Electroporation
  • NEPA21
  • Embryo
  • Zygote

This is a preview of subscription content, access via your institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-4939-9837-1_4
  • Chapter length: 11 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
eBook
USD   109.00
Price excludes VAT (USA)
  • ISBN: 978-1-4939-9837-1
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   149.99
Price excludes VAT (USA)
Hardcover Book
USD   219.99
Price excludes VAT (USA)
Fig. 1

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Singh P, Schimenti JC, Bolcun-Filas E (2015) A mouse geneticist’s practical guide to CRISPR applications. Genetics 199:1–15

    CAS  CrossRef  Google Scholar 

  2. Labun K, Montague TG, Gagnon JA, Thyme SB, Valen E (2016) CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering. Nucleic Acids Res 44:W272–W276

    CAS  CrossRef  Google Scholar 

  3. Concordet JP, Haeussler M (2018) CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Res 46:W242–WW45

    CAS  CrossRef  Google Scholar 

  4. Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, Smith I, Tothova Z, Wilen C, Orchard R, Virgin HW, Listgarten J, Root DE (2016) Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 34:184–191

    CAS  CrossRef  Google Scholar 

  5. Sentmanat MF, Peters ST, Florian CP, Connelly JP, Pruett-Miller SM (2018) A survey of validation strategies for CRISPR-Cas9 editing. Sci Rep 8:888

    CrossRef  Google Scholar 

  6. Kaneko T, Mashimo T (2015) Simple genome editing of rodent intact embryos by electroporation. PLoS One 10:e0142755

    CrossRef  Google Scholar 

  7. Anderson KR, Haeussler M, Watanabe C, Janakiraman V, Lund J, Modrusan Z, Stinson J, Bei Q, Buechler A, Yu C, Thamminana SR, Tam L, Sowick MA, Alcantar T, O’Neil N, Li J, Ta L, Lima L, Roose-Girma M, Rairdan X, Durinck S, Warming S (2018) CRISPR off-target analysis in genetically engineered rats and mice. Nat Methods 15:512–514

    CAS  CrossRef  Google Scholar 

  8. Luo C, Zuniga J, Edison E, Palla S, Dong W, Parker-Thornburg J (2011) Superovulation strategies for 6 commonly used mouse strains. J Am Assoc Lab Anim Sci 50:471–478

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Jay L. Vivian .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Verify currency and authenticity via CrossMark

Cite this protocol

Larson, M.A., Gibson, K.A., Vivian, J.L. (2020). In Vivo Validation of CRISPR Reagents in Preimplantation Mouse Embryos. In: Larson, M. (eds) Transgenic Mouse. Methods in Molecular Biology, vol 2066. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9837-1_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9837-1_4

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9836-4

  • Online ISBN: 978-1-4939-9837-1

  • eBook Packages: Springer Protocols