Skip to main content

Generation of Large Fragment Knock-In Mouse Models by Microinjecting into 2-Cell Stage Embryos

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

Abstract

Large fragment knock-in mouse models such as reporters and conditional mutant mice are important models for biological research. Here we describe 2-cell (2C)-homologous recombination (HR)-CRISPR, a highly efficient method to generate large fragment knock-in mouse models by CRISPR-based genome engineering. Using this method, knock-in founders can be generated routinely in a time frame of about two months with high germline transmission efficiency. 2C-HR-CRISPR will significantly promote the advancement of basic and translational research using genetic mouse models.

Key words

  • CRISPR-Cas9
  • 2-Cell stage
  • Knock-in
  • Homologous recombination

Dr. Posfai as Co-Corresponding Author

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_7
  • Chapter length: 12 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
Fig. 2
Fig. 3

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

References

  1. Yang H, Wang H, Jaenisch R (2014) Generating genetically modified mice using CRISPR/Cas-mediated genome engineering. Nat Protoc 9:1956–1968. https://doi.org/10.1038/nprot.2014.134

    CAS  CrossRef  PubMed  Google Scholar 

  2. Wang H et al (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153:910–918. https://doi.org/10.1016/j.cell.2013.04.025

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  3. Yang H et al (2013) One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154:1370–1379. https://doi.org/10.1016/j.cell.2013.08.022

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  4. Modzelewski AJ et al (2018) Efficient mouse genome engineering by CRISPR-EZ technology. Nat Protoc 13:1253–1274. https://doi.org/10.1038/nprot.2018.012

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  5. Qin W et al (2015) Efficient CRISPR/Cas9-mediated genome editing in mice by zygote electroporation of nuclease. Genetics 200:423–430. https://doi.org/10.1534/genetics.115.176594

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  6. Cohen J (2016) ‘Any idiot can do it.’ Genome editor CRISPR could put mutant mice in everyone’s reach. Science. https://doi.org/10.1126/science.aal0334

  7. Quadros RM et al (2017) Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. Genome Biol 18:92. https://doi.org/10.1186/s13059-017-1220-4

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  8. Yao X et al (2018) Tild-CRISPR allows for efficient and precise gene knockin in mouse and human cells. Dev Cell 45:526–536. e525. https://doi.org/10.1016/j.devcel.2018.04.021

    CAS  CrossRef  PubMed  Google Scholar 

  9. Nakade S et al (2014) Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9. Nat Commun 5:5560. https://doi.org/10.1038/ncomms6560

    CAS  CrossRef  PubMed  Google Scholar 

  10. Gu B, Posfai E, Rossant J (2018) Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos. Nat Biotechnol. https://doi.org/10.1038/nbt.4166

    CAS  CrossRef  PubMed  Google Scholar 

  11. Ma M et al (2017) Efficient generation of mice carrying homozygous double-floxp alleles using the Cas9-Avidin/biotin-donor DNA system. Cell Res 27:578–581. https://doi.org/10.1038/cr.2017.29

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  12. Ran FA et al (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8:2281–2308. https://doi.org/10.1038/nprot.2013.143

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  13. Behringer R, Gertsenstein M, Nagy K, Nagy A (2014) Manipulating the mouse embryo: a laboratory manual, 4th edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  14. Balakier H, Pedersen RA (1982) Allocation of cells to inner cell mass and trophectoderm lineages in preimplantation mouse embryos. Dev Biol 90:352–362

    CAS  CrossRef  PubMed  Google Scholar 

  15. Lawson KA, Pedersen RA (1987) Cell fate, morphogenetic movement and population kinetics of embryonic endoderm at the time of germ layer formation in the mouse. Development 101:627–652

    CAS  PubMed  Google Scholar 

  16. Wianny F, Zernicka-Goetz M (2000) Specific interference with gene function by double-stranded RNA in early mouse development. Nat Cell Biol 2:70–75. https://doi.org/10.1038/35000016

    CAS  CrossRef  PubMed  Google Scholar 

  17. Chazaud C, Yamanaka Y, Pawson T, Rossant J (2006) Early lineage segregation between epiblast and primitive endoderm in mouse blastocysts through the Grb2-MAPK pathway. Dev Cell 10:615–624. https://doi.org/10.1016/j.devcel.2006.02.020

    CAS  CrossRef  PubMed  Google Scholar 

  18. Swann K, Campbell K, Yu Y, Saunders C, Lai FA (2009) Use of luciferase chimaera to monitor PLCzeta expression in mouse eggs. Methods Mol Biol 518:17–29. https://doi.org/10.1007/978-1-59745-202-1_2

    CAS  CrossRef  PubMed  Google Scholar 

  19. Posfai E et al (2017) Position- and hippo signaling-dependent plasticity during lineage segregation in the early mouse embryo. Elife 6. https://doi.org/10.7554/eLife.22906

  20. Gertsenstein M, Nutter LMJ (2018) Engineering point putant and epitope-tagged alleles in mice using Cas9 RNA-guided nuclease. Curr Protoc Mouse Biol 8:28–53. https://doi.org/10.1002/cpmo.40

    CAS  CrossRef  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by CIHR (FDN-143334) and Genome Canada and Ontario Genomics (OGI-099). The authors wish to thank Dr. Janet Rossant for her guidance and support during the development of 2C-HR-CRISPR and critical discussion and comments.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Bin Gu .

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

Gu, B., Gertsenstein, M., Posfai, E. (2020). Generation of Large Fragment Knock-In Mouse Models by Microinjecting into 2-Cell Stage 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_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9837-1_7

  • 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