Advertisement

An Accessible Protocol for the Generation of CRISPR-Cas9 Knockouts Using INDELs in Zebrafish

  • Cara E. MoravecEmail author
  • Francisco J. Pelegri
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1920)

Abstract

The ability to create targeted mutations in specific genes, and therefore a loss-of-function condition, provides essential information about their endogenous functions during development and homeostasis. The discovery that CRISPR-Cas9 can target specific sequences according to base-pair complementarity and readily create knockouts in a desired gene has elevated the implementation of genetic analysis in numerous organisms. As CRISPR-Cas9 has become a powerful tool in a number of species, multiple methods for designing, creating, and screening editing efficiencies have been published, each of which has unique benefits. This chapter presents a cost-efficient, accessible protocol for creating knockout mutants in zebrafish using insertions/deletions (INDELS), from target site selection to mutant propagation, using basic laboratory supplies. The presented approach can be adapted to other systems, including any vertebrate species.

Key words

Genome editing CRISPR-Cas9 INDELS Targeted mutations Zebrafish 

References

  1. 1.
    Meng X, Noyes MB, Zhu LJ, Lawson ND, Wolfe SA (2008) Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Nat Biotechnol 26:695–701.  https://doi.org/10.1038/nbt1398 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Huang P, Xiao A, Zhou M, Zhu Z, Lin S, Zhang B (2011) Heritable gene targeting in zebrafish using customized TALENs. Nat Biotechnol 29:699–700.  https://doi.org/10.1038/nbt.1939 CrossRefPubMedGoogle Scholar
  3. 3.
    Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD et al (2013) Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 31:227–229.  https://doi.org/10.1038/nbt.2501 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA (2014) DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507:62–67.  https://doi.org/10.1038/nature13011 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Hwang WY, Fu Y, Reyon D, Maeder ML, Kaini P, Sander JD et al (2013) Heritable and precise zebrafish genome editing using a CRISPR-Cas system. PLoS One 8:e68708.  https://doi.org/10.1371/journal.pone.0068708 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Hisano Y, Sakuma T, Nakade S, Ohga R, Ota S, Okamoto H et al (2015) Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish. Sci Rep 5:8841.  https://doi.org/10.1038/srep08841 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Shankaran SS, Dahlem TJ, Bisgrove BW, Yost HJ, Tristani-Firouzi M (2017) CRISPR/Cas9-directed gene editing for the generation of loss-of-function mutants in high-throughput zebrafish F0 screens. Curr Protoc Mol Biol 119:31.9.1–31.9.22.  https://doi.org/10.1002/cpmb.42 CrossRefGoogle Scholar
  8. 8.
    Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP et al (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152:1173–1183.  https://doi.org/10.1016/j.cell.2013.02.022 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Chen B, Gilbert LA, Cimini BA, Schnitzbauer J, Zhang W, Li G-W et al (2013) Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155:1479–1491.  https://doi.org/10.1016/j.cell.2013.12.001 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Kearns NA, Pham H, Tabak B, Genga RM, Silverstein NJ, Garber M et al (2015) Functional annotation of native enhancers with a Cas9–histone demethylase fusion. Nat Methods 12:401–403.  https://doi.org/10.1038/nmeth.3325 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE et al (2013) CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154:442–451.  https://doi.org/10.1016/j.cell.2013.06.044 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Fernandez JP, Vejnar CE, Giraldez AJ, Rouet R, Moreno-Mateos MA (2018) Optimized CRISPR-Cpf1 system for genome editing in zebrafish. Methods.  https://doi.org/10.1016/j.ymeth.2018.06.014 CrossRefGoogle Scholar
  13. 13.
    Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J, Belanto JJ et al (2017) RNA targeting with CRISPR–Cas13. Nature 550:280.  https://doi.org/10.1038/nature24049 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    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.  https://doi.org/10.1093/nar/gkw398 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Montague TG, Cruz JM, Gagnon JA, Church GM, Valen E (2014) CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res 42:W401–W407.  https://doi.org/10.1093/nar/gku410 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Gagnon JA, Valen E, Thyme SB, Huang P, Ahkmetova L, Pauli A et al (2014) Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs. PLoS One 9:e98186.  https://doi.org/10.1371/journal.pone.0098186 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Zebrafish Microinjection Techniques | Protocol. https://www.jove.com/science-education/5130/zebrafish-microinjection-techniques. Accessed 1 Aug 2018.
  18. 18.
    Prykhozhij SV, Steele SL, Razaghi B, Berman JN (2017) A rapid and effective method for screening, sequencing and reporter verification of engineered frameshift mutations in zebrafish. Dis Model Mech 10:811–822.  https://doi.org/10.1242/dmm.026765 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V et al (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31:827–832.  https://doi.org/10.1038/nbt.2647 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Laboratory of GeneticsUniversity of Wisconsin–MadisonMadisonUSA

Personalised recommendations