Skip to main content
Book cover

Zebrafish pp 65–79Cite as

Generation of Targeted Genomic Deletions Through CRISPR/Cas System in Zebrafish

Part of the Methods in Molecular Biology book series (MIMB,volume 1451)

Abstract

Using TALEN or CRISPR/Cas system to induce small indels into coding sequences has been implicated in broad applications for reverse genetic studies of many organisms including zebrafish. However, complete deletion of a large gene or noncoding gene(s) or removing a large genomic fragment spanning several genes or other chromosomal elements is preferred in various cases, as well as inducing chromosomal inversions. Here, we describe the detailed protocols for the generation of chromosomal deletion mutations mediated by Cas9 and a pair of gRNAs and the evaluation for the efficiencies in F0 founder fish and of germline transmission.

Key words

  • Zebrafish
  • TALEN
  • CRISPR/Cas
  • Gene targeting
  • Genome manipulation
  • Gene disruption
  • Chromosomal deletion

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

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-4939-3771-4_5
  • Chapter length: 15 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: 978-1-4939-3771-4
  • 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   139.99
Price excludes VAT (USA)
Hardcover Book
USD   199.99
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. Huang P, Zhu Z, Lin S et al (2012) Reverse genetic approaches in zebrafish. J Genet Genomics 39(9):421–433. doi:10.1016/j.jgg.2012.07.004

    CAS  CrossRef  PubMed  Google Scholar 

  2. Xiao A, Wu Y, Yang Z et al (2013) EENdb: a database and knowledge base of ZFNs and TALENs for endonuclease engineering. Nucleic Acids Res 41(Database issue):D415–D422. doi:10.1093/nar/gks1144

    CAS  CrossRef  PubMed  Google Scholar 

  3. Hisano Y, Ota S, Kawahara A (2014) Genome editing using artificial site-specific nucleases in zebrafish. Dev Growth Differ 56(1):26–33. doi:10.1111/dgd.12094

    CAS  CrossRef  PubMed  Google Scholar 

  4. Bedell VM, Ekker SC (2015) Using engineered endonucleases to create knockout and knockin zebrafish models. Methods Mol Biol 1239:291–305. doi:10.1007/978-1-4939-1862-1_17

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  5. Xiao A, Wang Z, Hu Y et al (2013) Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish. Nucleic Acids Res 41(14):e141. doi:10.1093/nar/gkt464

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  6. Liu D, Wang Z, Xiao A et al (2014) Efficient gene targeting in zebrafish mediated by a zebrafish-codon-optimized cas9 and evaluation of off-targeting effect. J Genet Genomics 41(1):43–46. doi:10.1016/j.jgg.2013.11.004

    CrossRef  PubMed  Google Scholar 

  7. Ran FA, Hsu PD, Wright J et al (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8(11):2281–2308. doi:10.1038/nprot.2013.143

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  8. Hsu PD, Scott DA, Weinstein JA et al (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31(9):827–832. doi:10.1038/nbt.2647

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  9. Sander JD, Maeder ML, Reyon D, Voytas DF, Joung JK, Dobbs D (2010) ZiFiT (Zinc Finger Targeter): an updated zinc finger engineering tool. Nucleic Acids Res 38(Web Server issue):W462–W468. doi:10.1093/nar/gmk319

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  10. Xiao A, Cheng Z, Kong L et al (2014) CasOT: a genome-wide Cas9/gRNA off-target searching tool. Bioinformatics. doi:10.1093/bioinformatics/btt764

    Google Scholar 

  11. O’Brien A, Bailey TL (2014) GT-Scan: identifying unique genomic targets. Bioinformatics 30(18):2673–2675. doi:10.1093/bioinformatics/btu354

    CrossRef  PubMed  PubMed Central  Google Scholar 

  12. Huang P, Xiao A, Tong X et al (2014) TALEN construction via “Unit Assembly” method and targeted genome modifications in zebrafish. Methods 69(1):67–75. doi:10.1016/j.ymeth.2014.02.010

    CAS  CrossRef  PubMed  Google Scholar 

  13. Meeker ND, Hutchinson SA, Ho L et al (2007) Method for isolation of PCR-ready genomic DNA from zebrafish tissues. Biotechniques 43(5):610, 612, 614

    CrossRef  PubMed  Google Scholar 

  14. Zheng Q, Cai X, Tan MH et al (2014) Precise gene deletion and replacement using the CRISPR/Cas9 system in human cells. Biotechniques 57(3):115–124. doi:10.2144/000114196

    CAS  CrossRef  PubMed  Google Scholar 

  15. Cong L, Ran FA, Cox D et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819–823. doi:10.1126/science.1231143

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgment

We thank Zhanxiang Wang, Da Liu, and other members in our lab for their efforts on optimizing the CRISPR/Cas applications in zebrafish. This work was partially supported by the National Natural Science Foundation of China (31110103904, 81371264), the 973 Program of the Ministry of Science and Technology of China (2012CB945101, 2015CB942803), and the Seeding Grant for Medicine and Life Sciences of Peking University (2014-MB-06).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Bo Zhang .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Xiao, A., Zhang, B. (2016). Generation of Targeted Genomic Deletions Through CRISPR/Cas System in Zebrafish. In: Kawakami, K., Patton, E., Orger, M. (eds) Zebrafish. Methods in Molecular Biology, vol 1451. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3771-4_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-3771-4_5

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3769-1

  • Online ISBN: 978-1-4939-3771-4

  • eBook Packages: Springer Protocols